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PubMed1995

VISTAS: a package for VIsualizing STructures and sequences of proteins.

VISTAS is a suite of programs for protein sequence and structure analysis. The system allows the simultaneous display, in separate windows, of multiple sequence alignments, of known or model 3D structures, and of 2D graphic representations of sequence and/or alignment properties. The displays are fully integrated, and therefore manipulations in one window can be reflected in each of the others. Beyond its display facilities, VISTAS brings together a number of existing tools under a single, user-friendly umbrella: these include a fully functional interactive color alignment procedure, conserved motif selection, a range of database-scanning routines, and interactive access to the OWL composite sequence database and to the PRINTS protein fingerprint database. Exploration of the sequence database is thus straightforward, and predefined structural motifs from the fingerprint database may be readily visualized. Of particular note is the ability to calculate conservation criteria from sequence alignments and to display the information in a 3D context: this renders VISTAS a powerful tool for aiding mutagenesis studies and for facilitating refinement of molecular models.

Amino Acid Sequence↗
PubMed1996

A nested polymerase chain reaction for the detection of Borrelia burgdorferi sensu lato based on a multiple sequence analysis of the hbb gene.

A highly sensitive nested polymerase chain reaction method was designed for the detection of a wide spectrum of strains from Borrelia burgdorferi sensu lato. This technique allows the detection of as little as 3 fg of total genomic DNA extracted and purified from pure cultures of the organism, this amount corresponds to less than 10 organisms. Two sets of primers homologous to conserved spots in the coding region of the hbb gene, encoding a conserved histone-like protein, were constructed. These were based on a multiple sequence alignment of 39 strains representing all the genomic groups described in B. burgdorferi sensu lato.

Base Sequence↗
PubMed1993

Genomic sequences of murine gamma B- and gamma C-crystallin-encoding genes: promoter analysis and complete evolutionary pattern of mouse, rat and human gamma-crystallins.

The murine genes, gamma B-cry and gamma C-cry, encoding the gamma B- and gamma C-crystallins, were isolated from a genomic DNA library. The complete nucleotide (nt) sequences of both genes were determined from 661 and 711 bp, respectively, upstream from the first exon to the corresponding polyadenylation sites, comprising more than 2650 and 2890 bp, respectively. The new sequences were compared to the partial cDNA sequences available for the murine gamma B-cry and gamma C-cry, as well as to the corresponding genomic sequences from rat and man, at both the nt and predicted amino acid (aa) sequence levels. In the gamma B-cry promoter region, a canonical CCAAT-box, a TATA-box, putative NF-I and C/EBP sites were detected. An R-repeat is inserted 366 bp upstream from the transcription start point. In contrast, the gamma C-cry promoter does not contain a CCAAT-box, but some other putative binding sites for transcription factors (AP-2, UBP-1, LBP-1) were located by computer analysis. The promoter regions of all six gamma-cry from mouse, rat and human, except human psi gamma F-cry, were analyzed for common sequence elements. A complex sequence element of about 70-80 bp was found in the proximal promoter, which contains a gamma-cry-specific and almost invariant sequence (crygpel) of 14 nt, and ends with the also invariant TATA-box. Within the complex sequence element, a minimum of three further features specific for the gamma A-, gamma B- and gamma D/E/F-cry genes can be defined, at least two of which were recently shown to be functional. In addition to these four sequence elements, a subtype-specific structure of inverted repeats with different-sized spacers can be deduced from the multiple sequence alignment. A phylogenetic analysis based on the promoter region, as well as the complete exon 3 of all gamma-cry from mouse, rat and man, suggests separation of only five gamma-cry subtypes (gamma A-, gamma B-, gamma C-, gamma D- and gamma E/F-cry) prior to species separation.

Amino Acid Sequence↗
PubMed1994

Primary structure and biological features of a thermostable nuclease isolated from Staphylococcus hyicus.

The nucH gene, encoding a thermostable nuclease (TNase), was isolated from the cellular DNA of Staphylococcus hyicus strain E80 and sequenced. NucH, the 169-amino-acid (aa) protein encoded by this gene, contains, at its N-terminus, a signal peptide which appears to be cleaved at the same site in S. hyicus and Escherichia coli, yielding a mature protein which is exported extracellularly from S. hyicus, but not from E. coli. The aa sequence of NucH is highly homologous with that of the TNase from S. intermedius strain LRA076, whereas significant similarities are observed with the TNase from S. aureus, as well as with three other bacterial proteins of which only one has been shown to exhibit DNase activity. As seen in a multiple sequence alignment, the invariant residues are mostly located in the regions involved in the biological activity of the S. aureus TNase. The ability of crude cell extracts of E. coli strains carrying nucH to degrade various forms of nucleic acids with or without Ca2+ supplementation was studied. Under our experimental conditions, the enzyme encoded by nucH was active at 37 degrees C on both DNA and RNA, had the potential to act as an endonuclease, and functioned in the presence of Ca2+. Moreover, activity was retained after heating at 100 degrees C, suggesting that the enzyme could undergo reversible unfolding.

Amino Acid Sequence↗
PubMed1995

Isolation and characterization of pcp, a gene encoding a pyrrolidone carboxyl peptidase in Staphylococcus aureus.

The pcp gene, encoding a pyrrolidone carboxyl peptidase (PYRase), was cloned from a lambda GT11 genomic library prepared from Staphylococcus aureus FDA 574 and sequenced. The pcp gene is located 740 bp downstream from cna, a gene that encodes a collagen-binding adhesin in S. aureus. S. aureus pcp encodes a 212-amino-acid (aa) polypeptide. The pcp gene was overexpressed in Escherichia coli and the PYRase purified to homogeneity. The recombinant enzyme exhibited biological activity, as determined using the chromogenic substrate L-pyroglutamyl-beta-napthylamide. Biochemical analysis of the PYRase using thiol-blocking chemicals suggested that the enzyme belongs to the cysteine peptidase family. Moreover, multiple sequence alignment revealed a high degree of similarity to previously described bacterial PYRases. This family of peptidases has been used to selectively remove the N-terminal pyrrolidone carboxylic acid residue found on certain blocked proteins and peptides prior to aa sequencing. However, the exact biological role of PYRases has yet to be elucidated.

Amino Acid Sequence↗
PubMed1996

Cloning and characterisation of a gene from Plasmodium vivax and P. knowlesi: homology with valine-tRNA synthetase.

We have previously described a lambdagt11 clone detected by immune screening with a monoclonal antibody (mAb) A12. This mAb is capable of completely blocking Plasmodium vivax transmission in the mosquito vector. An epitope recognised by A12 was mapped to six amino acids (aa) within the translated sequence of this clone. Here, we describe the complete sequence of the gene within which we mapped this epitope. Surprisingly, the translated sequence of the full-length open reading frame shows homology with that of valine-tRNA synthetases (Val-tRS) from other organisms. DNA cross-hybridisation with several of these species was observed by Southern blot. In addition, the corresponding gene has been obtained from the closely related simian malaria parasite, P. knowlesi. The two aa sequences show 66% identity and yet are very divergent from other Val-tRS sequences, apart from conserved blocks related to functional activity. Multiple sequence alignments reflect this dichotomy, as do predicted differences in antigenicity.

Amino Acid Sequence↗
PubMed1994

Structure prediction of proteins--where are we now?

Although the 'structure from sequence' prediction problem remains fundamentally unsolved, new and promising methods in one, two and three dimensions have reopened the field. Significantly improved one-dimensional prediction of secondary structure from multiple sequence alignments is now in routine use. In the two-dimensional approach, inter-residue contacts can be detected by analysis of correlated mutations, albeit with low accuracy. Finally, three-dimensional methods, in which pseudopotentials or information values are derived from the databases, are proving their value for distinguishing between correct and incorrect models.

Amino Acid Sequence↗
PubMed1995

Characterization and modeling of membrane proteins using sequence analysis.

The current libraries of amino acid sequences of membrane proteins are a valuable resource for the analysis of elements common to these proteins. Multiple-sequence alignment techniques and the identification of conserved features of transmembrane segments have improved the prediction of membrane protein topology. Molecular modeling in combination with structural studies or site-directed mutagenesis is proving to be a powerful link between theory and experiment. Unfortunately, the number of high-resolution structures of intrinsic membrane proteins, although increased recently, presents a restricted and perhaps biased view of membrane protein structure.

Amino Acid Sequence↗
PubMed1993

Progress in protein structure prediction?

Prediction of protein secondary structure is an old problem and progress has been slow. Recently, spectacular success has been claimed in the blind prediction of the catalytic subunit of the cAMP-dependent protein kinase. When predictions in this and other test cases are assessed critically, some claims of prediction success turn out to be exaggerated, but a kernel of real progress remains: protein structure prediction can be improved substantially when a family of related sequences is available. Enough so that molecular biologists equipped with a new amino acid sequence and a multiple sequence alignment in hand may be tempted to test the new prediction methods.

Amino Acid Sequence↗
PubMed2005

Structure-activity relationship of an alpha-toxin Bs-Tx28 from scorpion (Buthus sindicus) venom suggests a new alpha-toxin subfamily.

Scorpion venoms are among the most widely known source of peptidyl neurotoxins used for callipering different ion channels, e.g., for Na(+), K(+), Ca(+) or Cl(-). An alpha-toxin (Bs-Tx28) has been purified from the venom of scorpion Buthus sindicus, a common yellow scorpion of Sindh, Pakistan. The primary structure of Bs-Tx28 was established using a combination of MALDI-TOF-MS, LC-ESI-MS, and automated Edman degradation analysis. Bs-Tx28 consists of 65 amino acid residues (7274.3+/-2Da), including eight cysteine residues, and shows very high sequence identity (82-94%) with other long-chain alpha-neurotoxins, active against receptor site-3 of mammalian (e.g., Lqq-IV and Lqh-IV from scorpions Leiurus sp.) and insect (e.g., BJalpha-IT and Od-1 from Buthotus judaicus and Odonthobuthus doriae, respectively) voltage-gated Na(+) channels. Multiple sequence alignment and phylogenetic analysis of Bs-Tx28 with other known alpha- and alpha-like toxins suggests the presence of a new and separate subfamily of scorpion alpha-toxins. Bs-Tx28 which is weakly active in both, mammals and insects (LD(50) 0.088 and 14.3microg/g, respectively), shows strong induction of the rat afferent nerve discharge in a dose-dependent fashion (EC(50)=0.01microg/mL) which was completely abolished in the presence of tetrodotoxin suggesting the binding of Bs-Tx28 to the TTX-sensitive Na(+)-channel. Three-dimensional structural features of Bs-Tx28, established by homology modeling, were compared with other known classical alpha-mammal (AaH-II), alpha-insect (Lqh-alphaIT), and alpha-like (BmK-M4) toxins and revealed subtle variations in the Nt-, Core-, and RT-CT-domains (functional domains) which constitute a "necklace-like" structure differing significantly in all alpha-toxin subfamilies. On the other hand, a high level of conservation has been observed in the conserved hydrophobic surface with the only substitution of W43 (Y43/42) and an additional hydrophobic character at position F40 (L40/A/V/G39), as compared to the other mentioned alpha-toxins. Despite major differences within the primary structure and activities of Bs-Tx28, it shares a common structural and functional motif (e.g., transRT-farCT) within the RT-CT domain which is characteristic of scorpion alpha-mammal toxins.

Action Potentials↗
PubMed2006

Cloning and functional analysis of the novel gene GhDBP3 encoding a DRE-binding transcription factor from Gossypium hirsutum.

A novel cDNA encoding DRE-binding transcription factor, designated GhDBP3, was cloned from Gossypium hirsutum. This protein was classified into A-4 group of DREB subfamily based on multiple sequence alignment and phylogenetic characterization. Semiquantitative RT-PCR showed that GhDBP3 was expressed in the leaves, cotyledons, roots and stems of 2-week-old cotton seedlings under non-stress conditions and was greatly induced in the cotton cotyledons by drought, NaCl, low temperature and ABA treatment. EMSA revealed that GhDBP3 was able to bind to the DRE cis-element in vitro. Transient assay using the particle bombardment method showed that GhDBP3 was a transcriptional activator, capable of activating expression of a reporter gene driven by the LEA D113 promoter containing a DRE like sequence in tobacco cells. Our results indicate that GhDBP3 could be a new member of DRE-binding transcription factor family and may play an important role in response to ABA and environmental stresses.

Amino Acid Sequence↗
PubMed2005

Identification of catalytically important amino acid residues of Streptomyces lividans acetylxylan esterase A from carbohydrate esterase family 4.

Multiple sequence alignment of Streptomyces lividans acetylxylan esterase A and other carbohydrate esterase family 4 enzymes revealed the following conserved amino acid residues: Asp-12, Asp-13, His-62, His-66, Asp-130, and His-155. These amino acids were mutated in order to investigate a functional role of these residues in catalysis. Replacement of the conserved histidine residues by alanine caused significant reduction of enzymatic activity. Maintenance of ionizable carboxylic group in side chains of amino acids at positions 12, 13, and 130 seems to be necessary for catalytic efficiency. The absence of conserved serine excludes a possibility that the enzyme is a serine esterase, in contrast to acetylxylan esterases of carbohydrate esterase families 1, 5, and 7. On the contrary, total conservation of Asp-12, Asp-13, Asp-130, and His-155 along with dramatic decrease in enzyme activity of mutants of either of these residues lead us to a suggestion that acetylxylan esterase A from Streptomyces lividans and, by inference, other members of carbohydrate esterase family 4 are aspartic deacetylases. We propose that one component of the aspartate dyad/triad functions as a catalytic nucleophile and the other one(s) as a catalytic acid/base. The ester/amide bond cleavage would proceed via a double displacement mechanism through covalently linked acetyl-enzyme intermediate of mixed anhydride type.

Acetylesterase↗
PubMed2003

Identification of a five-pass transmembrane protein family localizing in the Golgi apparatus and the ER.

A family of five-pass transmembrane proteins (FinGERs) were identified from the protein sequence database. The family includes yeast Yip1p, Yip4p, Yip5p, and Yif1p, and also their plant, insects, nematode, and mammalian homologues, suggesting their conserved function in a broad range of species. Eight family members were found in human. Multiple sequence alignment revealed three regions conserved among all family members. All of the human family members were expressed widely in various tissues. The human proteins were localized in and around the Golgi apparatus and may also be in the ER to some extent. The Golgi apparatus was fragmented by overexpression of the five of the family members. Some of the members were found to interact by yeast two-hybrid analysis, suggesting the formation of a complex. These results suggest that FinGERs function in maintenance of the Golgi structure and/or transport between the ER and the Golgi apparatus.

Amino Acid Sequence↗
PubMed2005

The HIN domain of IFI-200 proteins consists of two OB folds.

The interferon-inducible p200 (IFI-200/HIN-200) family of proteins regulates cell growth and differentiation, and confers resistance to the development of tumors and virus infections. IFI-200 family members are thought to exert their biological effects by modulation of the transcriptional activities of numerous factors and interaction with other proteins through the C-terminal HIN domains. However, the HIN domain structure and function have remained obscure. Therefore, we performed a comprehensive bioinformatics analysis and assembled a structure-based multiple sequence alignment of IFI-200 proteins. The application of fold recognition methods revealed that the HIN domain consists of two consecutive OB domains. Our structural models of DNA-binding HIN domains afford the long-sought interpretations for many previous experimental observations. Our results also raise the possibility of as yet unexplored functional roles of IFI-200 proteins as transcriptional regulators and as interaction partners of proteins involved in immunomodulatory and apoptotic processes.

Apoptosis↗
PubMed2006

Prediction of pi-turns in proteins using PSI-BLAST profiles and secondary structure information.

Due to the structural and functional importance of tight turns, some methods have been proposed to predict gamma-turns, beta-turns, and alpha-turns in proteins. In the past, studies of pi-turns were made, but not a single prediction approach has been developed so far. It will be useful to develop a method for identifying pi-turns in a protein sequence. In this paper, the support vector machine (SVM) method has been introduced to predict pi-turns from the amino acid sequence. The training and testing of this approach is performed with a newly collected data set of 640 non-homologous protein chains containing 1931 pi-turns. Different sequence encoding schemes have been explored in order to investigate their effects on the prediction performance. With multiple sequence alignment and predicted secondary structure, the final SVM model yields a Matthews correlation coefficient (MCC) of 0.556 by a 7-fold cross-validation. A web server implementing the prediction method is available at the following URL: http://210.42.106.80/piturn/.

Amino Acid Sequence↗
PubMed2006

Conformation and microenvironment of the active site of a low molecular weight 1,4-beta-D-glucan glucanohydrolase from an alkalothermophilic Thermomonospora sp.: involvement of lysine and cysteine residues.

Conformation and microenvironment at the active site of 1,4-beta-D-glucan glucanohydrolase was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde. OPTA has been known to form a fluorescent isoindole derivative by cross-linking the proximal thiol and amino groups of cysteine and lysine. Modification of lysine of the enzyme by TNBS and of cysteine residue by PHMB abolished the ability of the enzyme to form an isoindole derivative with OPTA. Kinetic analysis of the TNBS and PHMB-modified enzyme suggested the presence of essential lysine and cysteine residues, respectively, at the active site of the enzyme. The substrate protection of the enzyme with carboxymethylcellulose (CMC) confirmed the involvement of lysine and cysteine residues in the active site of the enzyme. Multiple sequence alignment of peptides obtained by tryptic digestion of the enzyme showed cysteine is one of the conserved amino acids corroborating the chemical modification studies.

Actinomycetales↗
PubMed2006

A new method for quantifying residue conservation and its applications to the protein folding nucleus.

The conservation of residues in columns of a multiple sequence alignment (MSA) reflects the importance of these residues for maintaining the structure and function of a protein. To date, many scores have been suggested for quantifying residue conservation, but none has achieved the full rigor both in biology and statistics. In this paper, we present a new approach for measuring the evolutionary conservation at aligned positions. Our conservation measure is related to the logarithmic probabilities for aligned positions, and combines the physicochemical properties and the frequencies of amino acids. Such a measure is both biologically and statistically meaningful. For testing the relationship between an amino acid's evolutionary conservation and its role in the Phi-value defined protein folding kinetics, our results indicate that the folding nucleus residues may not be significantly more conserved than other residues by using the biological-relevance weighted statistical scoring method suggested in this paper as an alternative to entropy-based procedures.

Amino Acid Sequence↗
PubMed2005

Characterization of Sparus aurata osteonectin cDNA and in silico analysis of protein conserved features: evidence for more than one osteonectin in Salmonidae.

Osteonectin is a matricellular protein involved in various cellular mechanisms but its exact function remains unclear despite numerous studies. We present here the cloning of Sparus aurata partial osteonectin cDNA and the reconstruction of 15 other sequences from both vertebrates and invertebrates, almost doubling the set of available sequences (a total of 35 sequences is now available). Taking advantage of the resulting large amount of data, we have created multiple sequence alignments and identified osteonectin putative conserved features (intra- and inter-disulfide bonds, collagen- and calcium-binding domains and phosphorylation sites) likely to be important for protein structure and function. This work also provides the first evidence for the presence of more than one osteonectin in some species. Finally, S. aurata osteonectin gene expression has been shown to initiate during larval development shortly after gastrulation, and to be high in bone-derived cell lines while down-regulated during extracellular matrix mineralization, further emphasizing the important role of osteonectin in skeletal development and bone formation.

Amino Acid Sequence↗