CEP: a conformational epitope prediction server.

CEP server (http://bioinfo.ernet.in/cep.htm) provides a web interface to the conformational epitope prediction algorithm developed in-house. The algorithm, apart from predicting conformational epitopes, also predicts antigenic determinants and sequential epitopes. The epitopes are predicted using 3D structure data of protein antigens, which can be visualized graphically. The algorithm employs structure-based Bioinformatics approach and solvent accessibility of amino acids in an explicit manner. Accuracy of the algorithm was found to be 75% when evaluated using X-ray crystal structures of Ag-Ab complexes available in the PDB. This is the first and the only method available for the prediction of conformational epitopes, which is an attempt to map probable antibody-binding sites of protein antigens.

VirGen: a comprehensive viral genome resource.

VirGen is a comprehensive viral genome resource that organizes the 'sequence space' of viral genomes in a structured fashion. It has been developed with the objective of serving as an annotated and curated database comprising complete genome sequences of viruses, value-added derived data and data mining tools. The current release (v1.1) contains 559 complete genomes in addition to 287 putative genomes of viruses belonging to eight viral families for which the host range includes animals and plants. Viral genomes in VirGen are annotated using sequence-based Bioinformatics approaches. The genomic data is also curated to identify 'alternate names' of viral proteins, where available. VirGen archives the results of comparisons of genomes, proteomes and individual proteins within and between viral species. It is the first resource to provide phylogenetic trees of viral species computed using whole-genome sequence data. The module of predicted B-cell antigenic determinants in VirGen is an attempt to link the genome to its vaccinome. Comparative genome analysis data facilitate the study of genome organization and evolution of viruses, which would have implications in applied research to identify candidates for the design of vaccines and antiviral drugs. VirGen is a relational database and is available at http://bioinfo. ernet.in/virgen/virgen.html.

Sequence alignment approach to pick up conformationally similar protein fragments.

Crystal structure data of globular proteins were used to prepare (phi, psi) probability maps of 20 proteinous amino acids. These maps were compared grid-wise with each other and a conformational similarity index was calculated for each pair of amino acids. A weight matrix, called Conformational Similarity Weight (CSW) matrix, was prepared using the conformational similarity index. This weight matrix was used to align sequences of 21 pairs of proteins whose crystal structures are known. The aligned regions with more than seven contiguous amino acids were further analysed by plotting average weight (W) values of overlapping hepatapeptides in these regions and carrying out curve fitting by Fourier series having TEN harmonics. The protein fragments corresponding to the half-linewidth of peaks were predicted as fragments having similar conformation in the protein pair under consideration. Such an approach allows us to pick up conformationally similar protein fragments with more than 67% accuracy.

Recognition of helper T cell epitopes in envelope (E) glycoprotein of Japanese encephalitis, west Nile and Dengue viruses.

Helper T (Th) cell antigenic sites were predicted from the primary amino acid sequence (approximately 500 in length) of the envelope (E) glycoprotein (gp) of Japanese encephalitis (JE), West Nile (WN) and Dengue (DEN) I-IV flaviviruses. Prediction of Th epitopes was done by analyzing the occurrence of amphipathic segments, Rothbard-Taylor tetra/pentamer motifs and presence of alpha helix-preferring amino acids. The simultaneous occurrence of all these parameters in segments of E gp were used as criteria for prediction as Th epitopes. Only one cross reactive epitope was predicted in the C-terminal region of the E gp predicted segments of all flaviviruses analyzed. This region is one of the longest amphipathic stretch (approximately from 420 to 455) and also has a fairly large amphipathic score. Based on the predicted findings three selected peptides were synthesized and analyzed for their ability to induce in vitro T cell proliferative response in different inbred strains of mice (Balb/c, C57BL6, C3H/HeJ). Synthetic peptide I and II prepared from C-terminal region gave a cross reactive response to JE, WN and Den-II in Balb/c and C3H/HeJ mice. Synthetic peptide III prepared from N-terminal region gave a proliferative response to DEN-II in Balb/c strain only, indicating differential antigen presentation.

Antigenic determinants reacting with rheumatoid factor: epitopes with different primary sequences share similar conformation.

Polyclonal or monoclonal human IgM rheumatoid factors (RF) react with eight antigenic sites on the CH3 IgG domain, four sites on CH2 and two on human beta 2-microglobulin. All 14 of these RF-reactive epitopes are linear 7-11 amino acid peptides with different primary sequence. We questioned whether RF reactivity with such a variety of epitopes showing no obvious sequence homology might result from conformational similarities shared by various RF-reactive regions. Strong support for this concept was obtained using rabbit antisera as well as mouse mAbs to individual CH3, CH2 or beta 2m RF-reactive peptides. Major cross-reactivity was demonstrated between most of the 14 different CH3, CH2, or beta 2m RF-reactive peptides using individual anti-epitope antibodies. Molecular modelling studies of these peptides showed striking similarities in three-dimensional shape among many RF-reactive peptides. Main-chain atoms rather than side chains seemed to contribute most directly to conformational similarity. Molecular simulation studies on control peptides showed no conformational similarities with RF-reactive peptides. Our studies indicate that autoantibodies such as RF recognize main-chain conformations of reactive epitopes and react with a number of antigenic determinants of quite different primary sequence but similar main chain conformations.

A semi-empirical method for prediction of antigenic determinants on protein antigens.

Analysis of data from experimentally determined antigenic sites on proteins has revealed that the hydrophobic residues Cys, Leu and Val, if they occur on the surface of a protein, are more likely to be a part of antigenic sites. A semi-empirical method which makes use of physicochemical properties of amino acid residues and their frequencies of occurrence in experimentally known segmental epitopes was developed to predict antigenic determinants on proteins. Application of this method to a large number of proteins has shown that our method can predict antigenic determinants with about 75% accuracy which is better than most of the known methods. This method is based on a single parameter and thus very simple to use.

Contextual constraints on codon pair usage: structural and biological implications.

Complementary DNA sequence data of 278 protein coding genes from prokaryotic systems have been analysed at the level of near neighbour codon pairs. Our analysis points out that constraints exist even at the level of near neighbour codon pairs. These constraints are in addition to those which arise due to relative levels of tRNA. Codon pairs, which in the data base have different occurrence values from their expected values, neither have common secondary structure nor do have better stabilization due to high base stacking. Our study points out that there are strong interaction between constituent codons in these codon pairs. These strongly interacting codon pairs, we suggest, are involved in the formation of three dimensional structural elements of cDNA/mRNA and interact with ribosome and thus modulate translation.

Molecular dynamics simulation of a 13-mer duplex DNA: a PvuII substrate.

Parallel version of AMBER 4.1 was ported and optimised on the Indian parallel supercomputer PARAM OpenFrame built around Sun Ultra Sparc processors. This version of AMBER program was then used to carry out molecular dynamics (MD) simulations on 5'-TGACCAGCTGGTC-3', a substrate for PvuII enzyme. MD simulations in water are carried out under following conditions: (i) unconstrained at 300 K (230 ps); (ii) unconstrained at 283 K (500 ps); (iii) Watson-Crick basepair constrained at 283 K (1 ns); and (iv) Watson-Crick basepair constrained with ions at 283 K (1.2 ns). In all these simulation studies, the molecule was observed to be bending and maximum distortions in the double helix around was seen around the G7:C7' basepair, which is the phosphodiester bond that is cleaved by PvuII. Analysis of MD simulation with ions carried out for 1.2 ns also pointed out that the conformation of double helix alternates between a conformation close to B-form and close to A-form. It is argued that a bent non-standard conformation is recognised by the PvuII enzyme. The maximum bend occurs at the G7:C7' region, weakening the phosphodiester bond and allows His48 to get placed in such a fashion to permit the scission through a general base mechanism. The bending and distortion observed is a property of the sequence which acts as a substrate for PvuII enzyme. This is confirmed by carrying out MD studies on the Dickerson's sequence d(CGCGAATTCGCG)2 as a reference molecule, which practically does not bend or get deformed.

Prediction of the recognition sites on 16S and 23S rRNAs from E. coli for the formation of 16S-23S rRNA complex.

Interactions between RNA molecules have been postulated to play an important role in the assembly of ribosomes. Using the sequence analysis and the search of continuous complementary regions on 16S rRNA and 23S rRNA, the recognition sites involved in the formation of ribosome of E. coli are postulated. The number of postulated sites was narrowed down by taking available experimental data. The suggestive evidence for correct postulation is obtained from sequence comparison studies of 16S and 23S rRNAs from various species. The sites 891-899 and 1195-1203 on 16S rRNA along with the corresponding complementary sites 1904-1912 and 760-768 on 23S rRNA are predicted to be the most probable candidates for the sites of recognition between 16S and 23S rRNAs. The possibility of the involvement of the additional site 630-638 on 16S rRNA with its complementary site 2031-2039 on 23S rRNA cannot be ruled out.

Online identification of viruses.

A computerized animal virus information system is developed in the Sequence Retrieval System (SRS) format. This database is available on the Word Wide Web (WWW) at the site http://bioinfo.ernet.in/www/avis/avis++ +.html. The database has been used to generate large number of identification matrices for each family. The software is developed in C. Unix shell scripts and Hypertext Marked-up Language (HTML) to assign the family to an unknown virus deterministically and to identify the virus probabilistically. It has been shown that such web based virus identification approach provides results with high confidence in those cases where identification matrix uses large number of independent characters. Protein sequence data for animal viruses have been analyzed and oligopeptides specific to each virus family and also specific to each virus species are identified for several viruses. These peptides thus could be used to identify the virus and to assign the virus family with high confidence showing the usefulness of sequence data in virus identification.

Obligatory amino acids in primitive proteins.

Conformational similarity among amino acid residues, a property derived by analysing (phi, psi)-probability distributions of 20 proteinous amino acids from 38 different globular proteins, is used to arrive at a set of six 'obligatory' amino acids of primitive proteins. The amino acids Ser, Val, Leu, Asp, Gly and Pro have been argued to be 'obligatory' and to represent, conformationally, the remaining amino acids. The reasons for consideration of these six residues as 'obligatory' are discussed. Methods to check the validity of our proposition are suggested.

Analysis of computer-predicted antibody inducing epitope on Japanese encephalitis virus.

Theoretical methods to delineate antibody inducing epitopes have been employed to predict antigenic determinants on envelope glycoprotein (gpE) of Japanese encephalitis (JE), West Nile (WN) and Dengue (DEN) I-IV viruses. A predicted region on JE virus gpE 74CPTTGEAHNEKRAD87 was synthesized, conjugated to KLH (KLH-peptide) and used in immunization of mice. A mouse monoclonal antibody (MoAb IVB4) reactive to the peptide was also found to react with native JE virus gpE. Characterization of the idiotypic (ID) determinants with the help of polyclonal domain-specific anti-ID antibodies revealed that polyclonal anti-KLH-peptide antibodies and MoAb IVB4 are flavivirus-cross-reactive to Hx and NHx domains, respectively. The region 74-87 in JE virus gpE has been mapped as a linking area between Hx and NHx domains. Reactivity of the peptide with sera from JE patients and vaccinees also indicated the feasibility of using predicted peptides for diagnostic and prophylastic purposes.

Contextual constraints in the choice of synonymous codons.

From EMBL Nucleotide Sequence Database, protein coding sequences of all E. coli and its DNA phages, were extracted using our computer programme. Same programme has been used to form a database of sequence of oligonucleotides of length 18 nucleotides on both sides of each of the 61 codons. From analysis of this database and study of variations in twist parameter (Tw) values, as an indicator of sequence dependent variations in B-DNA helix, a method is developed to fix the codon among the set of synonymous codons. The accuracy of the method was checked on enlarged data set by adding data from more prokaryotes. Our method assign the codon 85-90% times correctly if the selection has to be made between codons having different sequence in terms of R and Y. The accuracy of the method is somewhat lower when choice of the codon has to be made between codons having same codes in terms of R and Y. This study points out that the major factors which decide the choice of a codon from a set of synonymous codons are contextual constraints arising from flanking regions.

Categorization of metabolome in bacterial systems.

Analyses of biological databases such as those of genome, proteome, metabolome etc., have given insights in organization of biological systems. However, current efforts do not utilize the complete potential of available metabolome data. In this study, metabolome of bacterial systems with reliable annotations are analyzed and a simple method is developed to categorize pathways hierarchically, using rational approach. Ninety-four bacterial systems having for each ≥ 250 annotated metabolic pathways were used to identify a set of common pathways. 42 pathways were present in all bacteria which are termed as Core/Stage I pathways. This set of pathways was used along with interacting compounds to categorize pathways in the metabolome hierarchically. In each metabolome non-interacting pathways were identified including at each stage. The case study of Escherichia coli O157, having 433 annotated pathways, shows that 378 pathways interact directly or indirectly with 41 core pathways while 14 pathways are noninteracting. These 378 pathways are distributed in Stage II (289), Stage III (75), Stage IV (13) and Stage V (1) category. The approach discussed here allows understanding of the complexity of metabolic networks. It has pointed out that core pathways could be most ancient pathways and compounds that interact with maximum pathways may be compounds with high biosynthetic potential, which can be easily identified. Further, it was shown that interactions of pathways at various stages could be one to one, one to many, many to one or many to many mappings through interacting compounds. The granularity of the method discussed being high; the impact of perturbation in a pathway on the metabolome and particularly sub networks can be studied precisely. The categorizations of metabolic pathways help in identifying choke point enzymes that are useful to identify probable drug targets. The Metabolic categorizations for 94 bacteria are available at http://115.111.37.202/mpe/.

Towards a consensus on datasets and evaluation metrics for developing B-cell epitope prediction tools.

A B-cell epitope is the three-dimensional structure within an antigen that can be bound to the variable region of an antibody. The prediction of B-cell epitopes is highly desirable for various immunological applications, but has presented a set of unique challenges to the bioinformatics and immunology communities. Improving the accuracy of B-cell epitope prediction methods depends on a community consensus on the data and metrics utilized to develop and evaluate such tools. A workshop, sponsored by the National Institute of Allergy and Infectious Disease (NIAID), was recently held in Washington, DC to discuss the current state of the B-cell epitope prediction field. Many of the currently available tools were surveyed and a set of recommendations was devised to facilitate improvements in the currently existing tools and to expedite future tool development. An underlying theme of the recommendations put forth by the panel is increased collaboration among research groups. By developing common datasets, standardized data formats, and the means with which to consolidate information, we hope to greatly enhance the development of B-cell epitope prediction tools.

Computerization of virus data and its usefulness in virus classification.

Data on 537 Arboviruses and 180 other viruses have been collected and coded in two different formats. These data include information not only regarding the taxonomy and history of isolation, but also regarding the properties of biomacromolecules, proteins and nucleic acids. Information on antigenic relationships, histopathology and experimental viremia is also included. This information is stored in formats which allow the manipulation and analysis of data by dBASE III PLUS and MICRO-IS. A set of programs was written for interconversion and editing purposes. Transmission electron micrographs are scanned and stored. This stored information can be used in viral classification as shown by carrying out analysis of data on the Bunyaviridae family.

A method to locate protein coding sequences in DNA of prokaryotic systems.

cDNA sequence data from E. coli phages, for which complete genome sequences are known, have been analysed, From this analysis thirteen triplets have been identified as markers to distinguish protein-coding frames from fortuitous open reading frames. The region of -18 to +18 nucleotides around ATG/GTG, has been analysed and used to identify initiator codons from internal ATG/GTG. With the aid of criteria defined above a method has been developed to locate protein coding sequences by a combination of 'gene search by signal' and 'gene search by content' approaches. Application of this method to prokaryotic systems including those which were not part of our data base indicates that it is quite accurate and general in nature.