Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Multiple sequence alignment”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 451 records · Page 25Linked to original sources
PubMed1999

Molecular evolution of thyroid peroxidase.

Thyroid peroxidase is a member of a family of mammalian peroxidases that includes myeloperoxidase, lactoperoxidase, eosinophil peroxidase, and salivary peroxidase. Protein sequences showing a high degree of sequence similarity with mammalian peroxidases have recently been observed in several invertebrate species. A multiple sequence alignment prepared with five mammalian and six invertebrate peroxidases shows complete conservation of amino acid residues considered to be important in the formation of peroxidase compound 1. These include the distal and proximal histidines, a catalytic arginine residue, and an asparagine residue hydrogen bonded to the proximal histidine. TPO-2, an alternatively spliced form of TPO, lacks the essential asparagine (Asn 579). It is now possible to speak more broadly of the family of animal peroxidases, rather than mammalian peroxidases. The animal peroxidases comprise a group of homologous proteins that differ markedly from the plant/fungal/bacterial peroxidases in primary, secondary and tertiary structure, but which share with them a common function. Animal peroxidases probably arose independently of the plant/fungal/bacterial peroxidase superfamily and most likely belong to a different gene family. The relationship between animal and non-animal peroxidases probably represents an example of convergent evolution to a common enzymatic mechanism.

Amino Acid Sequence↗
PubMed2000

Oxygen transport proteins: III. Structural studies of the scorpion (Buthus sindicus) hemocyanin, partial primary structure of its subunit Bsin1.

The hemocyanin (Hc) from Buthus sindicus, studied in the native state, demonstrated to be an aggregate of eight different types of subunits arranged in four cubic hexamers. Both, the 'top' and the 'side' views of the native molecule have been identified from the negatively stained specimens using transmission electron microscopy. Out of these, eight different polypeptide chains, the partial primary structure (68%) of a subunit Bsin1 (Mr = 72422.7 Da) was established using a combination of automated Edman degradation and mass spectrometry. A multiple sequence alignment with other closely related cheliceratan Hc subunits revealed average identities of ca. 60%. Most of the structurally important residues, i.e. copper and calcium-binding ligands, as well as the residues involved in the presumed oxygen entrance pathway, proved to be strictly conserved in Bsin1. Sequence variations have been observed around the functionally important chloride-binding site, not only for the B. sindicus subunit Bsin1, but also for the subunit Aaus-6 of the scorpion A. australis and the subunit Ecal-a from the spider Eurypelma californicum Hcs. Deviation in the primary structure related to the chloride-binding site suggest that the effect of chloride ions may vary in different hemocyanins. Furthermore, the secondary structural contents of the Hc subunit Bsin1 were determined by circular dichroism revealing ca. 33% alpha-helix, 18%, beta-sheet, 19% beta-turn, and 30% random coil composition. These values are in good agreement with the crystal structure of the closely related Hc subunit Lpol-II from horseshoe crab L. polyphemus. Electron microscopic studies of the purified Hc subunit under native conditions revealed that Bsin1 has self aggregation properties. Results of these studies are discussed.

Amino Acid Sequence↗
PubMed2000

Structural similarities and evolutionary relationships in chloride-dependent alpha-amylases.

The alpha-amylase sequences contained in databanks were screened for the presence of amino acid residues Arg195, Asn298 and Arg/Lys337 forming the chloride-binding site of several specialized alpha-amylases allosterically activated by this anion. This search provides 38 alpha-amylases potentially binding a chloride ion. All belong to animals, including mammals, birds, insects, acari, nematodes, molluscs, crustaceans and are also found in three extremophilic Gram-negative bacteria. An evolutionary distance tree based on complete amino acid sequences was constructed, revealing four distinct clusters of species. On the basis of multiple sequence alignment and homology modeling, invariable structural elements were defined, corresponding to the active site, the substrate binding site, the accessory binding sites, the Ca(2+) and Cl(-) binding sites, a protease-like catalytic triad and disulfide bonds. The sequence variations within functional elements allowed engineering strategies to be proposed, aimed at identifying and modifying the specificity, activity and stability of chloride-dependent alpha-amylases.

Amino Acid Sequence↗
PubMed2000

Structural/functional assignment of unknown bacteriophage T4 proteins by iterative database searches.

Among the total of 274 orfs within bacteriophage T4, only half have been reasonably well characterized, and the functions of the rest have remained obscure. In order to predict the molecular functions of the orfs, a position-specific iterated (PSI)-BLAST search of bacteriophage T4 against the sequence database of known 3D structures was carried out. PSI-BLAST is one of the most powerful iterative sequence search methods using multiple sequence alignment, with the ability to detect many more proteins with distant homology than standard pairwise methods. The 3D structures of proteins are considered to be better preserved than the sequences, and the detected distantly homologous proteins are likely to possess highly similar 3D structures. Thirteen orfs of phage T4, whose homologues were not detected by standard pairwise methods, were found to have significantly homologous counterparts by this method. The plausibility of the results was confirmed by checking whether important residues at substrate/ligand-binding sites were conserved. Among them, two orfs, vs.1 and e.1, which are similar to Escherichia coli lytic enzyme and MutT protein, respectively, had not been studied previously. Also, gp rIIA, a rapid lysis protein, whose gene structure had been intensively studied during the development of molecular biology in the 1950s and yet whose molecular function remains unknown, has an N-terminal domain that is significantly similar to the N-terminal region of the heat shock protein Hsp90.

Amino Acid Sequence↗
PubMed2001

Cloning and expression of a nuclear encoded plastid specific 33 kDa ribonucleoprotein gene (33RNP) from pea that is light stimulated.

We report the cloning and sequencing of both cDNA and genomic DNA of a 33 kDa chloroplast ribonucleoprotein (33RNP) from pea. The analysis of the predicted amino acid sequence of the cDNA clone revealed that the encoded protein contains two RNA binding domains, including the conserved consensus ribonucleoprotein sequences CS-RNP1 and CS-RNP2, on the C-terminus half and the presence of a putative transit peptide sequence in the N-terminus region. The phylogenetic and multiple sequence alignment analysis of pea chloroplast RNP along with RNPs reported from the other plant sources revealed that the pea 33RNP is very closely related to Nicotiana sylvestris 31RNP and 28RNP and also to 31RNP and 28RNP of Arabidopsis and spinach, respectively. The pea 33RNP was expressed in Escherichia coli and purified to homogeneity. The in vitro import of precursor protein into chloroplasts confirmed that the N-terminus putative transit peptide is a bona fide transit peptide and 33RNP is localized in the chloroplast. The nucleic acid-binding properties of the recombinant protein, as revealed by South-Western analysis, showed that 33RNP has higher binding affinity for poly (U) and oligo dT than for ssDNA and dsDNA. The steady state transcript level was higher in leaves than in roots and the expression of this gene is light stimulated. Sequence analysis of the genomic clone revealed that the gene contains four exons and three introns. We have also isolated and analyzed the 5' flanking region of the pea 33RNP gene.

Amino Acid Sequence↗
PubMed1997

Identification of a novel member of the TGF-beta superfamily highly expressed in human placenta.

While conducting a gene discovery effort targeted to transcripts of the prevalent and intermediate frequency classes in placenta throughout gestation, we identified a novel member of the TGF-beta superfamily that is expressed at high levels in human placenta. Hence, we named this factor 'Placental Transforming Growth Factor Beta' (PTGFB). The full-length sequence of the 1.2-kb PTGFB mRNA has the potential of encoding a putative pre-pro-PTGFB protein of 295 amino acids and a putative mature PTGFB protein of 112 amino acids. Multiple sequence alignments of PTGFB and representative members of all TGF-beta subfamilies evidenced a number of conserved residues, including the seven cysteines that are almost invariant in all members of the TGF-beta superfamily. The single-copy PTGFB gene was shown to be composed of only two exons of 309 bp and 891 bp, separated by a 2.9-kb intron. The gene was localized to chromosome 19p12-13.1 by fluorescence in-situ hybridization. Northern analyses revealed a complex tissue-specific pattern of expression and a second transcript of 1.9 kb that is predominant in adult skeletal muscle. Most importantly, the 1.2-kb PTGFB transcript was shown to be expressed in placenta at much higher levels than in any other human fetal or adult tissue surveyed.

Adult↗
PubMed1999

Evolution of the proximal promoter region of the mammalian growth hormone gene.

The evolutionary relationship between the proximal growth hormone (GH) gene promoter sequences of 12 mammalian species was explored by comparison of their trinucleotide composition and by multiple sequence alignment. Both approaches yielded results that were consistent with the known fossil record-based phylogeny of the analysed sequences, suggesting that the two methods of tree reconstruction might be equally efficient and reliable. The pattern of evolution inferred for the mammalian GH gene promoters was found to vary both temporally and spatially. Thus, two distinct regions devoid of any evolutionary changes exist in primates, but only one of these 'gaps' is also observed in rodents, and neither is seen in ruminants. Furthermore, different evolutionary rates must have prevailed during different periods of evolutionary time and in different lineages, with a dramatic increase in evolutionary rate apparent in primates. Since a similar pattern of discontinuity has been previously noted for the evolution of the GH-coding regions, it may reflect the action of positive selection operating upon the GH gene as a single cohesive unit. Strong evidence for the action of gene conversion between primate GH gene promoters is provided by the fact that the human GH1 and GH2 sequences, which are thought to have diverged before the divergence of Old World monkeys from great apes, are more similar to one another than either is to the rhesus monkey GH2 promoter. Finally, it was noted that a number of nucleotide positions in the GH1 gene promoter that are polymorphic in humans appear to be highly conserved in mammals. This apparent conundrum, which could represent a caveat for the interpretation of phylogenetic footprinting studies, is potentially explicable in terms either of reduced genetic diversity in highly inbred animal species or insufficient population data from non-human species.

Animals↗
PubMed1999

Molecular phylogenetic analysis of felid herpesvirus 1.

The position of felid herpesvirus 1 within the alphaherpesvirus subfamily was investigated using molecular phylogenetic techniques applied to multiple sequence alignments of recently reported FHV-1 gene homologs (glycoprotein B, ribonucleotide reductase and DNA polymerase). FHV-1 was most closely related to other carnivore alphaherpesviruses, (phocid herpesvirus 1 and canid herpesvirus 1) and to the equid herpesviruses 1 and 4.

Alphaherpesvirinae↗
PubMed1998

Conserved motifs in T-cell receptor CDR1 and CDR2: implications for ligand and CD8 co-receptor binding.

Recent X-ray crystallographic structures of the T-cell receptor (TCR) alpha and beta chains, as well as their trimolecular complexes with peptide-MHC ligand, have established their structural similarity with the immunoglobulin molecules. The complementarity-determining region (CDR1) and CDR2 encoded within the TCR germline variable (V) sequence genes are well conserved across different TCR V alpha and V beta subfamilies. Multiple sequence alignments have been made based on structural information; they indicate that there will be only a limited number of canonical conformations for the first and second CDR loops. The limited diversity shown by CDRs 1 and 2 contrasts with the extreme junctional CDR3 diversity. Furthermore, CDR2 alignments have revealed conservation of a positive net charge in V alpha subfamilies. A model has been proposed for a direct interaction of the lateral part of CDR2 alpha with the negatively charged membrane-proximal 'stalk' region of the CD8 molecule.

Amino Acid Sequence↗
PubMed2002

Protein Explorer: easy yet powerful macromolecular visualization.

Protein Explorer (PE, http://www.proteinexplorer.org) enables students, educators and other nonspecialists to visualize macromolecular structures easily. It also offers several advanced capabilities useful to protein structure specialists. Great attention has been given to making PE easy to use. Explanations, color keys and troubleshooting information are displayed automatically. There are also 'Frequently Asked Questions', a one-hour 'Quick-Tour', an alphabetical 'Help/Index/Glossary', and a detailed 'Tutorial'; all making PE much easier to use than either Chime or RasMol. Moreover, it is much more powerful; in addition to basic macromolecular visualization capabilities common to most similar programs, it offers one-click visualization of interfaces between moieties ('contacts'), cation-pi interactions and salt bridges, as well as easy-to-use routines to visualize regions of conservation in three-dimensional protein structures based on multiple sequence alignments.

Computational Biology↗
PubMed2000

Interaction of pyrimethamine, cycloguanil, WR99210 and their analogues with Plasmodium falciparum dihydrofolate reductase: structural basis of antifolate resistance.

The nature of the interactions between Plasmodium falciparum dihydrofolate reductase (pfDHFR) and antimalarial antifolates, i.e., pyrimethamine (Pyr), cycloguanil (Cyc) and WR99210 including some of their analogues, was investigated by molecular modeling in conjunction with the determination of the inhibition constants (Ki). A three-dimensional structural model of pfDHFR was constructed using multiple sequence alignment and homology modeling procedures, followed by extensive molecular dynamics calculations. Mutations at amino acid residues 16 and 108 known to be associated with antifolate resistance were introduced into the structure, and the interactions of the inhibitors with the enzymes were assessed by docking and molecular dynamics for both wild-type and mutant DHFRs. The Ki values of a number of analogues tested support the validity of the model. A 'steric constraint' hypothesis is proposed to explain the structural basis of the antifolate resistance.

Amino Acid Sequence↗
PubMed1997

The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 A resolution: multiple copy flexibility and receptor binding.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic and vasculogenic mitogen. VEGF also plays a role in pathogenic vascularization which is associated with a number of clinical disorders, including cancer and rheumatoid arthritis. The development of VEGF antagonists, which prevent the interaction of VEGF with its receptor, may be important for the treatment of such disorders. VEGF is a homodimeric member of the cystine knot growth factor superfamily, showing greatest similarity to platelet-derived growth factor (PDGF). VEGF binds to two different tyrosine kinase receptors, kinase domain receptor (KDR) and Fms-like tyrosine kinase 1 (Flt-1), and a number of VEGF homologs are known with distinct patterns of specificity for these same receptors. The structure of VEGF will help define the location of the receptor-binding site, and shed light on the differences in specificity and cross-reactivity among the VEGF homologs. RESULTS: We have determined the crystal structure of the receptor-binding domain of VEGF at 1.93 A resolution in a triclinic space group containing eight monomers in the asymmetric unit. Superposition of the eight copies of VEGF shows that the beta-sheet core regions of the monomers are very similar, with slightly greater differences in most loop regions. For one loop, the different copies represent different snapshots of a concerted motion. Mutagenesis mapping shows that this loop is part of the receptor-binding site of VEGF. CONCLUSIONS: A comparison of the eight independent copies of VEGF in the asymmetric unit indicates the conformational space sampled by the protein in solution; the root mean square differences observed are similar to those seen in ensembles of the highest precision NMR structures. Mapping the receptor-binding determinants on a multiple sequence alignment of VEGF homologs, suggests the differences in specificity towards KDR and Flt-1 may derive from both sequence variation and changes in the flexibility of binding loops. The structure can also be used to predict possible receptor-binding determinants for related cystine knot growth factors, such as PDGF.

Amino Acid Sequence↗
PubMed1999

The structure of a Staphylococcus aureus leucocidin component (LukF-PV) reveals the fold of the water-soluble species of a family of transmembrane pore-forming toxins.

BACKGROUND: Leucocidins and gamma-hemolysins are bi-component toxins secreted by Staphylococcus aureus. These toxins activate responses of specific cells and form lethal transmembrane pores. Their leucotoxic and hemolytic activities involve the sequential binding and the synergistic association of a class S and a class F component, which form hetero-oligomeric complexes. The components of each protein class are produced as non-associated, water-soluble proteins that undergo conformational changes and oligomerization after recognition of their cell targets. RESULTS: The crystal structure of the monomeric water-soluble form of the F component of Panton-Valentine leucocidin (LukF-PV) has been solved by the multiwavelength anomalous dispersion (MAD) method and refined at 2.0 A resolution. The core of this three-domain protein is similar to that of alpha-hemolysin, but significant differences occur in regions that may be involved in the mechanism of pore formation. The glycine-rich stem, which undergoes a major rearrangement in this process, forms an additional domain in LukF-PV. The fold of this domain is similar to that of the neurotoxins and cardiotoxins from snake venom. CONCLUSIONS: The structure analysis and a multiple sequence alignment of all toxic components, suggest that LukF-PV represents the fold of any water-soluble secreted protein in this family of transmembrane pore-forming toxins. The comparison of the structures of LukF-PV and alpha-hemolysin provides some insights into the mechanism of transmembrane pore formation for the bi-component toxins, which may diverge from that of the alpha-hemolysin heptamer.

Amino Acid Sequence↗
PubMed1998

A combinatorial distance-constraint approach to predicting protein tertiary models from known secondary structure.

BACKGROUND: Distance geometry methods allow protein structures to be constructed using a large number of distance constraints, which can be elucidated by experimental techniques such as NMR. New methods for gleaning tertiary structural information from multiple sequence alignments make it possible for distance constraints to be predicted from sequence information alone. The basic distance geometry method can thus be applied using these empirically derived distance constraints. Such an approach, which incorporates a novel combinatoric procedure, is reported here. RESULTS: Given the correct sheet topology and disulfide formations, the fully automated procedure is generally able to construct native-like Calpha models for eight small beta-protein structures. When the sheet topology was unknown but disulfide connectivities were included, all sheet topologies were explored by the combinatorial procedure. Using a simple geometric evaluation scheme, models with the correct sheet topology were ranked first in four of the eight example cases, second in three examples and third in one example. If neither the sheet topology nor the disulfide connectivities were given a priori, all combinations of sheet topologies and disulfides were explored by the combinatorial procedure. The evaluation scheme ranked the correct topology within the top five folds for half the example cases. CONCLUSIONS: The combinatorial procedure is a useful technique for identifying a limited number of low-resolution candidate folds for small, disulfide-rich, beta-protein structures. Better results are obtained, however, if correct disulfide connectivities are known in advance. Combinatorial distance constraints can be applied whenever there are a sufficiently small number of finite connectivities.

Amino Acid Sequence↗
PubMed1995

An L40C mutation converts the cysteine-sulfenic acid redox center in enterococcal NADH peroxidase to a disulfide.

Multiple sequence alignments including the enterococcal NADH peroxidase and NADH oxidase indicate that residues Ser38 and Cys42 align with the two cysteines of the redox-active disulfides found in glutathione reductase (GR), lipoamide dehydrogenase, mercuric reductase, and trypanothione reductase. In order to evaluate those structural determinants involved in the selection of the cysteine-sulfenic acid (Cys-SOH) redox centers found in the two peroxide reductases and the redox-active disulfides present in the GR class of disulfide reductases, NADH peroxidase residues Ser38, Phe39, Leu40, and Ser41 have been individually replaced with Cys. Both the F39C and L40C mutant peroxidases yield active-site disulfides involving the new Cys and the native Cys42; formation of the Cys39-Cys42 disulfide, however, precludes binding of the FAD coenzyme. In contrast, the L40C mutant contains tightly-bound FAD and has been analyzed by both kinetic and spectroscopic approaches. In addition, the L40C and S41C mutant structures have been determined at 2.1 and 2.0 A resolution, respectively, by X-ray crystallography. Formation of the Cys40-Cys42 disulfide bond requires a movement of Cys42-SG to a new position 5.9 A from the flavin-C(4a) position; this is consistent with the inability of the new disulfide to function as a redox center in concert with the flavin. Stereochemical constraints prohibit formation of the Cys41-Cys42 disulfide in the latter mutant.

Amino Acid Sequence↗
PubMed1992

A proteolytically sensitive region common to several rat liver cytochromes P450: effect of cleavage on substrate binding.

Limited proteolysis of rat liver microsomes was used to probe the topography and structure of cytochrome P450 bound to the endoplasmic reticulum. Three cytochromes P450 from two families were examined. Monoclonal antibodies to cytochrome P450 forms 1A1, 2B1, and 2E1 were used to immunopurify these proteolyzed cytochromes P450 from microsomes from rats treated with 3-methylcholanthrene, phenobarbital, and acetone, respectively. Electrophoretic and immunoblot analysis of tryptic fragments revealed a highly sensitive cleavage site in all three cytochromes P450. N-Terminal sequencing was performed on the fragments after transfer onto poly(vinylidene difluoride) membranes and showed that this preferential cleavage site is at amino acid position 298 of P450 1A1, position 277 of P450 2B1, and position 278 of P450 2E1. Multiple sequence alignment revealed that these positions are at the amino terminal of a highly conserved region of these cytochromes P450. The important functional role implied by primary sequence conservation along with the proteolytic sensitivity at its amino terminal suggests that this region is a protein domain. Comparison with the known structure of the bacterial cytochrome P450cam predicts that this proteolytically sensitive site is within an interhelical turn region connected to the distal helix that partially encompasses the heme-containing active site. Substrate binding to the cleaved cytochromes P450 was examined in order to determine whether the newly added conformational freedom near the cleavage site functionally altered these cytochromes P450. Cleavage of P450 2B1 abolished benzphetamine binding, which indicates that the cleavage site contains an important structural determinant for binding this substrate. However, cleavage did not affect benzo[a]pyrene binding to P450 1A1.

Amino Acid Sequence↗
PubMed2000

Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase.

Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates.

Amino Acid Sequence↗
PubMed2001

Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: site-directed mutagenesis of highly conserved residues.

Dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes alkylation of the exocyclic amine of adenosine at position 37 in some tRNAs by the hydrocarbon moiety of dimethylallyl diphosphate (DMAPP). A multiple-sequence alignment of 28 gene sequences encoding DMAPP-tRNA transferases from various organisms revealed considerable homology, including 11 charged, 12 polar, and four aromatic amino acids that are highly conserved or conservatively substituted. Site-directed mutants were constructed for all of these amino acids, and a tripeptide Glu-Glu-Phe alpha-tubulin epitope was appended to the C-terminus of the protein to facilitate separation by immunoaffinity chromatography of overproduced mutant enzymes from coexpressed chromosomally encoded wild-type DMAPP-tRNA transferase. Steady-state kinetic constants were measured for wild-type DMAPP-tRNA transferase and the site-directed mutants using DMAPP and a 17-base RNA oligoribonucleotide corresponding to the stem-loop region of tRNA(Phe) as substrates. Substantial changes in k(cat), K(m)(DMAPP), and/or K(m)(RNA) were seen for several of the mutants, suggesting possible roles for these residues in substrate binding and catalysis.

Alkyl and Aryl Transferases↗