Orthogonal beta-beta motifs in proteins

A super-secondary structural motif comprising two orthogonally oriented beta-strands connected by short linking segments of <5 residues has been identified from a data set of 65 independent protein crystal structures. Of the 42 examples from 14 proteins, a vast majority have only a single residue as the linking element. Analysis of the conformational angles at the junction reveals that the recently described type VIII beta-turn occurs frequently at the connecting hinge, while the type II beta-turn is also fairly common.

Use of protein A gene fusions for the analysis of structure-function relationship of the transactivator protein C of bacteriophage Mu

A sensitive dimerization assay for DNA binding proteins has been developed using gene fusion technology. For this purpose, we have engineered a gene fusion using protein A gene of Staphylococcus aureus and C gene, the late gene transactivator of bacteriophage Mu. The C gene was fused to the 3' end of the gene for protein A to generate an A- C fusion. The overexpressed fusion protein was purified in a single step using immunoglobulin affinity chromatography. Purified fusion protein exhibits DNA binding activity as demonstrated by electrophoretic mobility shift assays. When the fusion protein A-C was mixed with C and analyzed for DNA binding, in addition to C and A-C specific complexes, a single intermediate complex comprising of a heterodimer of C and A-C fusion proteins was observed. Further, the protein A moiety in the fusion protein A-C does not contribute to DNA binding as demonstrated by proteolytic cleavage and circular dichroism (CD) analysis. The assay has also been applied to analyze the DNA binding domain of C protein by generating fusions between protein A and N- and C-terminal deletion mutants of C. The results indicate a role for the region towards the carboxy terminal of the protein in DNA binding. The general applicability of this method is discussed.

Analysis of the amino acid sequences of plant Bowman-Birk inhibitors

Plant seeds contain a large number of protease inhibitors of animal, fungal, and bacterial origin. One of the well-studied families of these inhibitors is the Bowman-Birk family(BBI). The BBIs from dicotyledonous seeds are 8K, double-headed proteins. In contrast, the 8K inhibitors from monocotyledonous seeds are single headed. Monocots also have a 16K, double-headed inhibitor. We have determined the primary structure of a Bowman-Birk inhibitor from a dicot, horsegram, by sequential edman analysis of the intact protein and peptides derived from enzymatic and chemical cleavage. The 76-residue-long inhibitor is very similar to that ofMacrotyloma axillare. An analysis of this inhibitor along with 26 other Bowman-Birk inhibitor domains (MW 8K) available in the SWISSPROT databank revealed that the proteins from monocots and dicots belong to related but distinct families. Inhibitors from monocots show larger variation in sequence. Sequence comparison shows that a crucial disulphide which connects the amino and carboxy termini of the active site loop is lost in monocots. The loss of a reactive site in monocots seems to be correlated to this. However, it appears that this disulphide is not absolutely essential for retention of inhibitory function. Our analysis suggests that gene duplication leading to a 16K inhibitor in monocots has occurred, probably after the divergence of monocots and dicots, and also after the loss of second reactive site in monocots.

Arabidopsis thaliana J-class heat shock proteins: cellular stress sensors

Plants are sessile organisms that have evolved a variety of mechanisms to maintain their cellular homeostasis under stressful environmental conditions. Survival of plants under abiotic stress conditions requires specialized group of heat shock protein machinery, belonging to Hsp70:J-protein family. These heat shock proteins are most ubiquitous types of chaperone machineries involved in diverse cellular processes including protein folding, translocation across cell membranes, and protein degradation. They play a crucial role in maintaining the protein homeostasis by reestablishing functional native conformations under environmental stress conditions, thus providing protection to the cell. J-proteins are co-chaperones of Hsp70 machine, which play a critical role by stimulating Hsp70s ATPase activity, thereby stabilizing its interaction with client proteins. Using genome-wide analysis of Arabidopsis thaliana, here we have outlined identification and systematic classification of J-protein co-chaperones which are key regulators of Hsp70s function. In comparison with Saccharomyces cerevisiae model system, a comprehensive domain structural organization, cellular localization, and functional diversity of A. thaliana J-proteins have also been summarized. Electronic supplementary material The online version of this article (doi:10.1007/s10142-009-0132-0) contains supplementary material, which is available to authorized users.

Pi-turns in proteins and peptides: Classification, conformation, occurrence, hydration and sequence.

The i + 5-->i hydrogen bonded turn conformation (pi-turn) with the fifth residue adopting alpha L conformation is frequently found at the C-terminus of helices in proteins and hence is speculated to be a "helix termination signal." An analysis of the occurrence of i + 5-->i hydrogen bonded turn conformation at any general position in proteins (not specifically at the helix C-terminus), using coordinates of 228 protein crystal structures determined by X-ray crystallography to better than 2.5 A resolution is reported in this paper. Of 486 detected pi-turn conformations, 367 have the (i + 4)th residue in alpha L conformation, generally occurring at the C-terminus of alpha-helices, consistent with previous observations. However, a significant number (111) of pi-turn conformations occur with (i + 4)th residue in alpha R conformation also, generally occurring in alpha-helices as distortions either at the terminii or at the middle, a novel finding. These two sets of pi-turn conformations are referred to by the names pi alpha L and pi alpha R-turns, respectively, depending upon whether the (i + 4)th residue adopts alpha L or alpha R conformations. Four pi-turns, named pi alpha L'-turns, were noticed to be mirror images of pi alpha L-turns, and four more pi-turns, which have the (i + 4)th residue in beta conformation and denoted as pi beta-turns, occur as a part of hairpin bend connecting twisted beta-strands. Consecutive pi-turns occur, but only with pi alpha R-turns. The preference for amino acid residues is different in pi alpha L and pi alpha R-turns. However, both show a preference for Pro after the C-termini. Hydrophilic residues are preferred at positions i + 1, i + 2, and i + 3 of pi alpha L-turns, whereas positions i and i + 5 prefer hydrophobic residues. Residue i + 4 in pi alpha L-turns is mainly Gly and less often Asn. Although pi alpha R-turns generally occur as distortions in helices, their amino acid preference is different from that of helices. Poor helix formers, such as His, Tyr, and Asn, also were found to be preferred for pi alpha R-turns, whereas good helix former Ala is not preferred. pi-Turns in peptides provide a picture of the pi-turn at atomic resolution. Only nine peptide-based pi-turns are reported so far, and all of them belong to pi alpha L-turn type with an achiral residue in position i + 4. The results are of importance for structure prediction, modeling, and de novo design of proteins.

Strategies for efficient disruption of metabolism in Mycobacterium tuberculosis from network analysis

Tuberculosis continues to be a major health challenge, warranting the need for newer strategies for therapeutic intervention and newer approaches to discover them. Here, we report the identification of efficient metabolism disruption strategies by analysis of a reactome network. Protein-protein dependencies at a genome scale are derived from the curated metabolic network, from which insights into the nature and extent of inter-protein and inter-pathway dependencies have been obtained. A functional distance matrix and a subsequent nearness index derived from this information, helps in understanding how the influence of a given protein can pervade to the metabolic network. Thus, the nearness index can be viewed as a metabolic disruptability index, which suggests possible strategies for achieving maximal metabolic disruption by inhibition of the least number of proteins. A greedy approach has been used to identify the most influential singleton, and its combination with the other most pervasive proteins to obtain highly influential pairs, triplets and quadruplets. The effect of deletion of these combinations on cellular metabolism has been studied by flux balance analysis. An obvious outcome of this study is a rational identification of drug targets, to efficiently bring down mycobacterial metabolism.

Nonspecifically bound proteins spin while diffusing along DNA

It is known that DNA-binding proteins can slide along the DNA helix while searching for specific binding sites, but their path of motion remains obscure. Do these proteins undergo simple one-dimensional (1D) translational diffusion, or do they rotate to maintain a specific orientation with respect to the DNA helix? We measured 1D diffusion constants as a function of protein size while maintaining the DNA-protein interface. Using bootstrap analysis of single-molecule diffusion data, we compared the results to theoretical predictions for pure translational motion and rotation-coupled sliding along the DNA. The data indicate that DNA-binding proteins undergo rotation-coupled sliding along the DNA helix and can be described by a model of diffusion along the DNA helix on a rugged free-energy landscape. A similar analysis including the 1D diffusion constants of eight proteins of varying size shows that rotation-coupled sliding is a general phenomenon. The average free-energy barrier for sliding along the DNA was 1.1 +/- 0.2 k(B)T. Such small barriers facilitate rapid search for binding sites.

Structure-Based Phylogeny as a Diagnostic for Functional Characterization of Proteins with a Cupin Fold

Background: The members of cupin superfamily exhibit large variations in their sequences, functions, organization of domains, quaternary associations and the nature of bound metal ion, despite having a conserved beta-barrel structural scaffold. Here, an attempt has been made to understand structure-function relationships among the members of this diverse superfamily and identify the principles governing functional diversity. The cupin superfamily also contains proteins for which the structures are available through world-wide structural genomics initiatives but characterized as ``hypothetical''. We have explored the feasibility of obtaining clues to functions of such proteins by means of comparative analysis with cupins of known structure and function. Methodology/Principal Findings: A 3-D structure-based phylogenetic approach was undertaken. Interestingly, a dendrogram generated solely on the basis of structural dissimilarity measure at the level of domain folds was found to cluster functionally similar members. This clustering also reflects an independent evolution of the two domains in bicupins. Close examination of structural superposition of members across various functional clusters reveals structural variations in regions that not only form the active site pocket but are also involved in interaction with another domain in the same polypeptide or in the oligomer. Conclusions/Significance: Structure-based phylogeny of cupins can influence identification of functions of proteins of yet unknown function with cupin fold. This approach can be extended to other proteins with a common fold that show high evolutionary divergence. This approach is expected to have an influence on the function annotation in structural genomics initiatives.

CsrA interacting small RNAs in Haemophilus spp genomes: a theoretical analysis

The csrA is a carbon storage regulator gene that encodes a protein with multiple RNA interaction sites. Bacterial non-coding small RNAs like csrB, csrC and their counterparts in diverse bacterial genus are identified to control the regulatory activities of CsrA and its orthologs. An attempt has been made in this study to identify 'novel' non-coding small RNAs that are involved in the regulatory activities of csrA gene. All CsrA-interacting small RNAs are computationally fingerprinted to have multiple occurrence of 7-nucleotide CsrA interacting repeats [CAGGA(U/A/C)G] along with a 18-nucleotide upstream binding site. However, in several of the genomes like Haemophilus spp, the upstream binding site is not identified. The current methodology overcomes this difficulty by identifying small RNA-specific orphan transcriptional units within the intergenic regions of the genome. The results could identify all known CsrA-interacting small RNAs in E. coli, Vibrio cholerae and Pseudomonas aeruginosa genomes and additionally has picked six new possible CsrA-interacting small RNA regions in E. coli. Our computational analysis indicates that known rygD and rprA sRNAs in E. coli could possibly interact with CsrA proteins. The study is extended to three of the Haemophilus genomes that could identify seven new possible CsrA interacting small RNAs.

Computational analysis of proteome of H5N1 avian influenza virus to define T cell epitopes with vaccine potential

The existing vaccines against influenza are based on the generation of neutralizing antibody primarily directed against surface proteins-hernagglutinin and neuraminidase. In this work, we have computationally defined conserved T cell epitopes of proteins of influenza virus H5N1 to help in the design of a vaccine with haplotype specificity for a target population. The peptides from the proteome of H5NI irus which are predicted to bind to different HLAs, do not show similarity with peptides of human proteorne and are also identified to be generated by proteolytic cleavage. These peptides could be made use of in the design of either a DNA vaccine or a subunit vaccine against V influenza. (c) 2007 Elsevier Ltd. All rights reserved.

Rotavirus nonstructural proteins: a structural perspective

Rotavirus is a major cause of acute infantile diarrhoea worldwide. The virus genome consists of 11 segments of double-stranded RNA that codesfor six structural proteins (VP1-6) and six non-structural proteins(NSP1-6). NSPs are proteins expressed from the virus genome in the infected cell, but are not incorporated into the mature virus article. NSPs play an essential role in virus replication, morphogenesis and pathogenesis, and most of them exhibit multifunctional properties. Structure-function analysis of the NSPs is essential for understanding the molecular mechanisms by which the virus circumvents host innate immune responses, inhibits cellular protein synthesis, hijacks the protein synthetic machinery for its own propagation and manifests the disease process. Because of their essential roles in virus biology, NSPs represent potential targets for the development of antiviral agents. Determination of the three-dimensional structure of NSPs has been hindered due to low-level expression and aggregation. To date, the complete three-dimensional structure of only NSP2 has been determined. The structures of the N- and C-terminal domains of NSP3 and the diarrhoea-inducing domain of NSP4 have also been determined. This review primarily covers the structural and biological functions of the NSPs whose three-dimensional structural aspects have been fully or partially understood, but provides a brief account of other NSPs and the structural features of the mature virion as determined by electron cryomicroscopy.

Conformations of disulfide bridges in proteins

The conformational characteristics of disulfide bridges in proteins have been analyzed using a dataset of 22 protein structures, available at a resolution of 2.0 Å, containing a total of 72 disulfide crosslinks. The parameters used in the analysis include (φ, Ψ) values at Cys residues, bridge dihedral angles χss, χ1i, χ1j, χ2i and χ2j the distances Cαi-Cαj and Cβi-Cβj between the Cα and Cβ atoms of Cys(i) and Cys(j). Eight families of bridge conformations with three or more occurrences have been identified on the basis of these stereochemical parameters. The most populated family corresponds to the "left handed spiral" identified earlier by Richardson ((1981) Adv. Protein Chem. 34, 167–330). Disulfide bridging across antiparallel extended strands is observed in α-lytic protease, crambin, and β-trypsin and this structure is shown to be very similar to those obtained in small cystine peptides. Solvent accessible surface area calculations show that the overwhelming majority of disulfide bridges are inaccessible to solvent.

Conformational characteristics of asparaginyl residues in proteins

Backbone conformations at 1064 asparaginyl residues in 123 non-homologous, high-resolution X-ray structures of proteins were analysed. Asn adopts conformations in left-handed x-helical region and other partially allowed regions in the Ramachandran map more readily than any other non-glycyl residue. Asn conformational clusters in the (phi,psi) regions of left-handed alpha-helix, right-handed alpha-helix and extended (beta) strands were investigated in detail for their occurrence in various secondary structures, especially in beta-turn regions. Preferences were observed for Asn conformations in different positions in various beta-turn types, including the first and fourth positions of the turn. Asparaginyl residues with extended conformations are found to occur frequently in irregular regions, although they are expected to occur predominantly in extended strands or in the third position of type II beta-turns. Asn conformations at the N-cap positions of helices strongly prefer extended conformation than alpha(L), which seems to be characteristic of non-glycyl residues at that position. In the linkers connecting two extended strands and those connecting an alpha-helix and an extended strand, Asn with alpha(L) or alpha(R) conformation is more favoured than Asn with the beta-conformation. Analysis of Asn-Asn doublets and Asn-X-Asn triplets permitted identification of conformational families in such sequences. Results of this investigation provide useful hints in modelling Asn-rich regions in proteins such as malaria parasite coat protein. (C) Munksgaard 1994.

Protein Block Expert (PBE): a web-based protein structure analysis server using a structural alphabet

Encoding protein 3D structures into 1D string using short structural prototypes or structural alphabets opens a new front for structure comparison and analysis. Using the well-documented 16 motifs of Protein Blocks (PBs) as structural alphabet, we have developed a methodology to compare protein structures that are encoded as sequences of PBs by aligning them using dynamic programming which uses a substitution matrix for PBs. This methodology is implemented in the applications available in Protein Block Expert (PBE) server. PBE addresses common issues in the field of protein structure analysis such as comparison of proteins structures and identification of protein structures in structural databanks that resemble a given structure. PBE-T provides facility to transform any PDB file into sequences of PBs. PBE-ALIGNc performs comparison of two protein structures based on the alignment of their corresponding PB sequences. PBE-ALIGNm is a facility for mining SCOP database for similar structures based on the alignment of PBs. Besides, PBE provides an interface to a database (PBE-SAdb) of preprocessed PB sequences from SCOP culled at 95% and of all-against-all pairwise PB alignments at family and superfamily levels. PBE server is freely available at http://bioinformatics.univ-reunion.fr/ PBE/.

Lectin Microarrays for Glycomic Analysis

Glycomics is the study of comprehensive structural elucidation and characterization of all glycoforms found in nature and their dynamic spatiotemporal changes that are associated with biological processes. Glycocalyx of mammalian cells actively participate in cell-cell, cell-matrix, and cell-pathogen interactions, which impact embryogenesis, growth and development, homeostasis, infection and immunity, signaling, malignancy, and metabolic disorders. Relative to genomics and proteomics, glycomics is just growing out of infancy with great potential in biomedicine for biomarker discovery, diagnosis, and treatment. However, the immense diversity and complexity of glycan structures and their multiple modes of interactions with proteins pose great challenges for development of analytical tools for delineating structure function relationships and understanding glycocode. Several tools are being developed for glycan profiling based on chromatography,m mass spectrometry, glycan microarrays, and glyco-informatics. Lectins, which have long been used in glyco-immunology, printed on a microarray provide a versatile platform for rapid high throughput analysis of glycoforms of biological samples. Herein, we summarize technological advances in lectin microarrays and critically review their impact on glycomics analysis. Challenges remain in terms of expansion to include nonplant derived lectins, standardization for routine clinical use, development of recombinant lectins, and exploration of plant kingdom for discovery of novel lectins.