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PubMed2003

Increased coverage obtained by combination of methods for protein sequence database searching.

MOTIVATION: Sequence alignment methods that compare two sequences (pairwise methods) are important tools for the detection of biological sequence relationships. In genome annotation, multiple methods are often run and agreement between methods taken as confirmation. In this paper, we assess the advantages of combining search methods by comparing seven pairwise alignment methods, including three local dynamic programming algorithms (PRSS, SSEARCH and SCANPS), two global dynamic programming algorithms (GSRCH and AMPS) and two heuristic approximations (BLAST and FASTA), individually and by pairwise intersection and union of their result lists at equal p-value cut-offs. RESULTS: When applied singly, the dynamic programming methods SCANPS and SSEARCH gave significantly better coverage (p=0.01) compared to AMPS, GSRCH, PRSS, BLAST and FASTA. Results ranked by BLAST p-values gave significantly better coverage compared to ranking by BLAST e-values. Of 56 combinations of eight methods considered, 19 gave significant increases in coverage at low error compared to the parent methods at an equal p-value cutoff. The union of results by BLAST (p-value) and FASTA at an equal p-value cutoff gave significantly better coverage than either method individually. The best overall performance was obtained from the intersection of the results from SSEARCH and the GSRCH62 global alignment method. At an error level of five false positives, this combination found 444 true positives, a significant 12.4% increase over SSEARCH applied alone.

Algorithms↗

PubMed2000

The C-terminal domain of pancreatic lipase: functional and structural analogies with c2 domains.

The 3D structure of pancreatic lipase (PL) consists of two functional domains. The N-terminal domain belongs to the alpha/beta hydrolase fold and contains the active site, which involves a catalytic triad analogous to that present in serine proteases. The beta-sandwich C-terminal domain of PL plays an important part in the binding process between the lipase and colipase, the specific PL cofactor. Recent structure-function studies have suggested that the PL C-terminal domain may have an extra role apart from that of binding colipase. This domain contains an exposed hydrophobic loop (beta5') which was found to be located on the same side as the hydrophobic loops surrounding the active site, and it may be involved in the lipid binding process. Indirect evidence for this new function of the PL C-terminal domain has been provided by studies with monoclonal antibodies directed against the beta5' loop. The catalytic activity of the PL-antibody complexes on water insoluble substrates decreased drastically, whereas their esterase activity on a soluble substrate remained unchanged. During the last few years, a number of protein structures (15-lipoxygenase, alpha-toxin from Clostridium perfringens) have been determined that contain domains with close structural homologies with the beta-sandwich C-terminal domain of PL. Generally speaking, these domains show structural homologies with the C2 domains occurring in a wide range of proteins involved in signal transduction (e.g. phosphoinositide-specific phospholipase C, protein kinase C, cytosolic phospholipase A2), membrane traffic (e.g. synaptotagmin I, rabphilin) and membrane disruption (e.g. perforin). Here it is proposed to review the structure and function of the C2 domains, based on the recent 3D structures and improved sequence alignments.

Amino Acid Sequence↗

PubMed1998

Combined multiple sequence reduced protein model approach to predict the tertiary structure of small proteins.

By incorporating predicted secondary and tertiary restraints into ab initio folding simulations, low resolution tertiary structures of a test set of 20 nonhomologous proteins have been predicted. These proteins, which represent all secondary structural classes, contain from 37 to 100 residues. Secondary structural restraints are provided by the PHD secondary structure prediction algorithm that incorporates multiple sequence information. Predicted tertiary restraints are obtained from multiple sequence alignments via a two-step process: First, "seed" side chain contacts are identified from a correlated mutation analysis, and then, the seed contacts are "expanded" by an inverse folding algorithm. These predicted restraints are then incorporated into a lattice based, reduced protein model. Depending upon fold complexity, the resulting nativelike topologies exhibit a coordinate root-mean-square deviation, cRMSD, from native between 3.1 and 6.7 A. Overall, this study suggests that the use of restraints derived from multiple sequence alignments combined with a fold assembly algorithm is a promising approach to the prediction of the global topology of small proteins.

Algorithms↗

PubMed2004

Exon-intron structure of outlier tick lipocalins indicate a monophyletic origin within the larger lipocalin family.

All tick proteins assigned to the lipocalin family lack the structural conserved regions (SCRs) that are characteristic of the kernel lipocalins and can thus be classified as outliers. These tick proteins have been assigned to the tick lipocalin family based on database searches that indicated homology between tick sequences and the fact that the histamine binding protein (HBP2) from the hard tick Rhipicephalus appendiculatus (Ixodidae) shows structural similarity to the lipocalin fold. Sequence identity between kernel and outlier lipocalins falls below 20% and the question raised is whether the outlier and kernel lipocalins are truly homologous. More specifically in the case of the tick lipocalins, whether their structural fold is derived from the lipocalin fold or whether convergent evolution resulted in the generation of the basic lipocalin-like fold which consists of an eight stranded continuous anti-parallel beta-barrel terminated by a C-terminal alpha-helix that lies parallel to the barrel. The current study determined the gene structure for HBP2 and TSGP1, TSGP2 and TSGP4, lipocalins identified from the soft tick Ornithodoros savignyi (Argasidae). All tick lipocalins have four introns (A-D) with conserved positions and phases within the tick lipocalin sequence alignment. The positions and phase information are also conserved with regard to the rest of the lipocalin family. Phylogenetic analysis using this information shows conclusively that tick lipocalins are evolutionary related to the rest of the lipocalin family. Tick lipocalins are grouped within a monophyletic clade that indicates a monophyletic origin within the tick lineage and also group with the other arthropod lipocalins in a larger clade. Phylogenetic analysis of sequence alignments based on conserved secondary structure of the lipocalin fold support the conclusions from the gene structure trees. These results indicate that exon-intron arrangement can be useful for the inclusion of outlier lipocalins within the larger lipocalin family.

Amino Acid Sequence↗

PubMed2005

Structure conservation in cytochromes P450.

The recent availability of crystal structures for several diverse cytochromes P450 (CYPs) offers the possibility to perform an up-to-date comparative analysis to identify the degree of structure conservation among this superfamily of enzymes specially relevant for their involvement in drug metabolism and toxicity. A set of 9 CYPs sharing between 10% and 27% sequence identity was selected, including 7 class I (CYP 101, 107, 108, 119, 121, 51, and 55) and two class II (CYP 102, and 2C5) structures. After obtaining a multiprotein structure superimposition, a structure-based sequence alignment was derived. Mapping the level of three-dimensional structural conservation onto the sequence alignment revealed that over 28% of the alignment positions have the Calpha carbons of their residues within a root-mean-square deviation (RMSD) of 2 A. This degree of structure conservation is found to be generally preserved, even when the structure undergoes dramatic conformational changes. Performing the analysis on 4 members of the CYP2 family (CYP 2B4, 2C5, 2C8, and 2C9), the percentage of alignment positions within 2 A RMSD amounted to 73%, increasing to over 85% when only structures in a closed conformation are considered. The present findings suggest that it should be plausible to derive models of overall good quality for the major CYP2 metabolizing forms (CYP 2A6, 2C19, 2D6, and 2E1), whereas high levels of uncertainty are still likely to be expected in models for the remaining 2 major P450 metabolizing forms (CYP 1A2 and 3A4), with the corresponding implications for their potential applicability in drug design activities.

Amino Acid Motifs↗

PubMed2002

A sliding window-based method to detect selective constraints in protein-coding genes and its application to RNA viruses.

Here we present a new sliding window-based method specially designed to detect selective constraints in specific regions of a multiple protein-coding sequence alignment. In contrast to previous window-based procedures, our method is based on a nonarbitrary statistical approach to find the appropriate codon-window size to test deviations of synonymous (d(S)) and nonsynonymous (d(N)) nucleotide substitutions from the expectation. The probabilities of d(N) and d(S) are obtained from simulated data and used to detect significant deviations of d(N) and d(S) in a specific window region of the real sequence alignment. The nonsynonymous-to-synonymous rate ratio (w = d(N)/d(S)) was used to highlight selective constraints in any window wherein d(S) or d(N) was significantly different from the expectation. In these significant windows, w and its variance [V(w)] were calculated and used to test the neutral hypothesis. Computer simulations showed that the method is accurate even for highly divergent sequences. The main advantages of the new method are that it (i) uses a statistically appropriate window size to detect different selective patterns, (ii) is computationally less intensive than maximum likelihood methods, and (iii) detects saturation of synonymous sites, which can give deviations from neutrality. Hence, it allows the analysis of highly divergent sequences and the test of different alternative hypothesis as well. The application of the method to different human immunodeficiency virus type 1 and to foot-and-mouth disease virus genes confirms the action of positive selection on previously described regions as well as on new regions.

Base Sequence↗

PubMed2000

Evolution of genes and taxa: a primer.

The rapidly growing fields of molecular evolution and systematics have much to offer to molecular biology, but like any field have their own repertoire of terms and concepts. Homology, for example, is a central theme in evolutionary biology whose definition is complex and often controversial. Homology extends to multigene families, where the distinction between orthology and paralogy is key. Nucleotide sequence alignment is also a homology issue, and is a key stage in any evolutionary analysis of sequence data. Models based on our understanding of the processes of nucleotide substitution are used both in the estimation of the number of evolutionary changes between aligned sequences and in phylogeny reconstruction from sequence data. The three common methods of phylogeny reconstruction--parsimony, distance and maximum likelihood--differ in their use of these models. All three face similar problems in finding optimal--and reliable--solutions among the vast number of possible trees. Moreover, even optimal trees for a given gene may not reflect the relationships of the organisms from which the gene was sampled. Knowledge of how genes evolve and at what rate is critical for understanding gene function across species or within gene families. The Neutral Theory of Molecular Evolution serves as the null model of molecular evolution and plays a central role in data analysis. Three areas in which the Neutral Theory plays a vital role are: interpreting ratios of nonsynonymous to synonymous nucleotide substitutions, assessing the reliability of molecular clocks, and providing a foundation for molecular population genetics.

Base Sequence↗

PubMed1999

Combining many multiple alignments in one improved alignment.

MOTIVATION: The fact that the multiple sequence alignment problem is of high complexity has led to many different heuristic algorithms attempting to find a solution in what would be considered a reasonable amount of computation time and space. Very few of these heuristics produce results that are guaranteed always to lie within a certain distance of an optimal solution (given a measure of quality, e.g. parsimony). Most practical heuristics cannot guarantee this, but nevertheless perform well for certain cases. An alignment, obtained with one of these heuristics and with a bad overall score, is not unusable though, it might contain important information on how substrings should be aligned. This paper presents a method that extracts qualitatively good sub-alignments from a set of multiple alignments and combines these into a new, often improved alignment. The algorithm is implemented as a variant of the traditional dynamic programming technique. RESULTS: An implementation of ComAlign (the algorithm that combines multiple alignments) has been run on several sets of artificially generated sequences and a set of 5S RNA sequences. To assess the quality of the alignments obtained, the results have been compared with the output of MSA 2.1 (Gupta et al., Proceedings of the Sixth Annual Symposium on Combinatorial Pattern Matching, 1995; Kececioglu et al., http://www.techfak.uni-bielefeld. de/bcd/Lectures/kececioglu.html, 1995). In all cases, ComAlign was able to produce a solution with a score comparable to the solution obtained by MSA. The results also show that ComAlign actually does combine parts from different alignments and not just select the best of them. AVAILABILITY: The C source code (a Smalltalk version is being worked on) of ComAlign and the other programs that have been implemented in this context are free and available on WWW (http://www.daimi.au.dk/ õcaprani). CONTACT: klaus@bucka-lassen.dk; jotun@pop.bio.au.dk;ocaprani@daimi.au.dk

Algorithms↗

PubMed1993

Evolutionary relationships among the serpins.

The serpins are a widely distributed group of serine proteinase inhibitors found in plants, birds, mammals and viruses. Despite the great evolutionary divergence of these organisms, their serpins are highly conserved, both in sequence and structurally. Amino acid sequences were aligned by a combination of automatic algorithms and by consideration of conserved structural elements in those serpins for which crystal structures exist. The program HOMED was used which allowed the alignment of amino acids to be simultaneously converted into the equivalently aligned nucleotide sequences. The aligned amino acids were used as the basis for superposition of the four known three-dimensional structures for which coordinates are available and compared with an optimal three-dimensional superposition in order to estimate the reliability of the sequence alignment. Phylogenetic relationships implied by these nucleotide sequence alignments were determined by the method of maximum parsimony. The proposed gene tree suggested that as much diversity existed between the plant serpin and mammalian serpins as was present among mammalian serpins and provided further evidence that the architecture of serpin molecules is highly constrained.

Amino Acid Sequence↗

PubMed2005

Norovirus classification and proposed strain nomenclature.

Without a virus culture system, genetic analysis becomes the principal method to classify norovirus (NoV) strains. Currently, classification of NoV strains beneath the species level has been based on sequences from different regions of the viral genome. As a result, the phylogenetic insights of some virus were not appropriately interpreted, and no consensus has been reached to establish a uniform classification scheme. To provide a consistent and reliable scientific basis for classifying NoVs, we analyzed the amino acid sequences for the major capsid protein of 164 NoV strains by first using an alignment based on the predicted 3D structures. A Bayesian tree was generated, and the maximum likelihood pairwise distances of the aligned sequences were used to evaluate the results from the uncorrected pairwise distance method. Analyses of the pairwise distances demonstrated three clearly resolved peaks, suggesting that NoV strains beneath the species level can be classified at three levels: strain (S), cluster (C), and genogroup (G). The uncorrected pairwise distance ranges for S, C, and G were 0-14.1%, 14.3-43.8%, and 44.9-61.4%, respectively. A scheme with 29 genetic clusters [8 in genogroup 1 (G1), 17 in G2, 2 in G3, and 1 each in G4 and G5] was defined on the basis of the tree topology with the standards provided and was supported by the distance analysis. Of these, five clusters in G2 and one in G1 are newly described. This analysis can serve as the basis for a standardized nomenclature to genetically describe NoV strains.

Animals↗

PubMed1997

Nucleotide sequences and gene organization of TaqI endonuclease isoschizomers from Thermus sp. SM32 and Thermus filiformis Tok6A1.

Eight TaqI isoschizomer genes, two from Yellowstone National Park, one from Japan, two from New Zealand, two from Portugal, and one from the Azores (1000 miles west of Portugal), were PCR-amplified and sequenced. Sequence alignment of isoschizomers isolated from close geographical locations shows identical or almost identical protein sequences, while isoschizomers from distant sites demonstrate considerable diversity, ranging from 54 to 75% in amino acid identity. Accordingly, these isoschizomers were arranged into four geographical groups, i.e., USA as represented by Thermus aquaticus YT1, Japan by Thermus thermophilus HB8, New Zealand by Thermus filiformis Tok6A1, Portugal by Thermus sp. SM32. The complete ORFs of two new representative genes, tfiTok6A1I and tsp32IR, were obtained by bubble PCR. Unlike M . TaqI-R.TaqI and M . TthHB8I-R . TthHB8I which exhibit an unusual 13-codon overlap, the methylase and endonuclease genes are each separated by 15 nucleotides in the TfiTok6A1I and Tsp32IR restriction-modification systems. Phylogenetic analysis suggests that initially TfiTok6A1I diverged from a common ancestor, then Tsp32IR branched out, and finally TaqI and TthHB8I diverged from each other during evolution.

Amino Acid Sequence↗

PubMed1993

Identification and classification of protein fold families.

We have developed a method for identifying fold families in the protein structure data bank. Pairwise sequence alignments are first performed to extract families of homologous proteins having 35% or more sequence identity. Representatives are selected with the best resolution and R-factor to give a nonhomologous data set. Subsequent structure comparisons between all members of this set detect homologous folds with low sequence identity but highly conserved structures. By softening the requirement on structural similarity, families of analogous proteins are obtained that have related folds but more diverse structures. Representatives are selected to give a non-analogous data set. Starting with 1410 chains from the Brookhaven Data Bank, we generate a set of 150 nonhomologous folds and a set of 112 non-analogous folds. Analysis of sequence and structure conservation within the larger families shows the globins to be the most highly conserved family and the TIM barrels the most weakly conserved.

Classification↗

PubMed2001

Characterization of the hepatitis C virus NS2/3 processing reaction by using a purified precursor protein.

The NS2-NS3 region of the hepatitis C virus polyprotein encodes a proteolytic activity that is required for processing of the NS2/3 junction. Membrane association of NS2 and the autocatalytic nature of the NS2/3 processing event have so far constituted hurdles to the detailed investigation of this reaction. We now report the first biochemical characterization of the self-processing activity of a purified NS2/3 precursor. Using multiple sequence alignments, we were able to define a minimal domain, devoid of membrane-anchoring sequences, which was still capable of performing the processing reaction. This truncated protein was efficiently expressed and processed in Escherichia coli. The processing reaction could be significantly suppressed by growth in minimal medium in the absence of added zinc ions, leading to the accumulation of an unprocessed precursor protein in inclusion bodies. This protein was purified to homogeneity, refolded, and shown to undergo processing at the authentic NS2/NS3 cleavage site with rates comparable to those observed using an in vitro-translated full-length NS2/3 precursor. Size-exclusion chromatography and a dependence of the processing rate on the concentration of truncated NS2/3 suggested a functional multimerization of the precursor protein. However, we were unable to observe trans cleavage activity between cleavage-site mutants and active-site mutants. Furthermore, the cleavage reaction of the wild-type protein was not inhibited by addition of a mutant that was unable to undergo self-processing. Site-directed mutagenesis data and the independence of the processing rate from the nature of the added metal ion argue in favor of NS2/3 being a cysteine protease having Cys993 and His952 as a catalytic dyad. We conclude that a purified protein can efficiently reproduce processing at the NS2/3 site in the absence of additional cofactors.

Amino Acid Sequence↗

PubMed1986

The classification of amino acid conservation.

A classification of amino acid type is described which is based on a synthesis of physico-chemical and mutation data. This is organised in the form of a Venn diagram from which sub-sets are derived that include groups of amino acids likely to be conserved for similar structural reasons. These sets are used to describe conservation in aligned sequences by allocating to each position the smallest set that contains all the residue types brought together by alignment. This minimal set assignment provides a simple way of reducing the information contained in a sequence alignment to a form which can be analysed by computer yet remains readable.

Amino Acids↗

PubMed2005

A pairwise alignment algorithm which favors clusters of blocks.

Pairwise sequence alignments aim to decide whether two sequences are related and, if so, to exhibit their related domains. Recent works have pointed out that a significant number of true homologous sequences are missed when using classical comparison algorithms. This is the case when two homologous sequences share several little blocks of homology, too small to lead to a significant score. On the other hand, classical alignment algorithms, when detecting homologies, may fail to recognize all the significant biological signals. The aim of the paper is to give a solution to these two problems. We propose a new scoring method which tends to increase the score of an alignment when "blocks" are detected. This so-called Block-Scoring algorithm, which makes use of dynamic programming, is worth being used as a complementary tool to classical exact alignments methods. We validate our approach by applying it on a large set of biological data. Finally, we give a limit theorem for the score statistics of the algorithm.

Algorithms↗

PubMed2001

The relationship between the L1 and L2 domains of the insulin and epidermal growth factor receptors and leucine-rich repeat modules.

BACKGROUND: Leucine-rich repeats are one of the more common modules found in proteins. The leucine-rich repeat consensus motif is LxxLxLxxNxLxxLxxLxxLxx- where the first 11-12 residues are highly conserved and the remainder of the repeat can vary in size Leucine-rich repeat proteins have been subdivided into seven subfamilies, none of which include members of the epidermal growth factor receptor or insulin receptor families despite the similarity between the 3D structure of the L domains of the type I insulin-like growth factor receptor and some leucine-rich repeat proteins. RESULTS: Here we have used profile searches and multiple sequence alignments to identify the repeat motif Ixx-LxIxx-Nx-Lxx-Lxx-Lxx-Lxx- in the L1 and L2 domains of the insulin receptor and epidermal growth factor receptors. These analyses were aided by reference to the known three dimensional structures of the insulin-like growth factor type I receptor L domains and two members of the leucine rich repeat family, porcine ribonuclease inhibitor and internalin 1B. Pectate lyase, another beta helix protein, can also be seen to contain the sequence motif and much of the structural features characteristic of leucine-rich repeat proteins, despite the existence of major insertions in some of its repeats. CONCLUSION: Multiple sequence alignments and comparisons of the 3D structures has shown that right-handed beta helix proteins such as pectate lyase and the L domains of members of the insulin receptor and epidermal growth factor receptor families, are members of the leucine-rich repeat superfamily.

Amino Acid Sequence↗

PubMed2003

Comparison methods for branching and axillary flowering sequences.

Comparing branching and axillary flowering patterns accurately is a major issue both in botany and in various agronomic contexts. Data take the form of sequences which naturally represent the underlying structural information of branching and axillary flowering patterns. Various comparison methods are proposed based either on sequence alignment or on the computation of dissimilarity measures between (hidden) Markovian models built from sets of sequences. Sequence alignment is a natural complement to the exploratory tools and statistical models proposed in Guédon et al. (J. Theor. Biol. 212 (2001) 481) with the distinctive feature of applying to individual sequences. Comparison methods may also be used to reveal some grouping within a set of sequences or to evaluate the strength of a predefined grouping of sequences. The proposed approach is illustrated by examples corresponding to different plant species and different biological or agronomic objectives.

Computer Simulation↗

PubMed1991

Eukaryotic DNA polymerase amino acid sequence required for 3'----5' exonuclease activity.

We have identified an amino-proximal sequence motif, Phe-Asp-Ile-Glu-Thr, in Saccharomyces cerevisiae DNA polymerase II that is almost identical to a sequence comprising part of the 3'----5' exonuclease active site of Escherichia coli DNA polymerase I. Similar motifs were identified by amino acid sequence alignment in related, aphidicolin-sensitive DNA polymerases possessing 3'----5' proofreading exonuclease activity. Substitution of Ala for the Asp and Glu residues in the motif reduced the exonuclease activity of partially purified DNA polymerase II at least 100-fold while preserving the polymerase activity. Yeast strains expressing the exonuclease-deficient DNA polymerase II had on average about a 22-fold increase in spontaneous mutation rate, consistent with a presumed proofreading role in vivo. In multiple amino acid sequence alignments of this and two other conserved motifs described previously, five residues of the 3'----5' exonuclease active site of E. coli DNA polymerase I appeared to be invariant in aphidicolin-sensitive DNA polymerases known to possess 3'----5' proofreading exonuclease activity. None of these residues, however, appeared to be identifiable in the catalytic subunits of human, yeast, or Drosophila alpha DNA polymerases.

Amino Acid Sequence↗