Progressive structure-based alignment of homologous proteins: Adopting sequence comparison strategies

Comparison of multiple protein structures has a broad range of applications in the analysis of protein structure, function and evolution. Multiple structure alignment tools (MSTAs) are necessary to obtain a simultaneous comparison of a family of related folds. In this study, we have developed a method for multiple structure comparison largely based on sequence alignment techniques. A widely used Structural Alphabet named Protein Blocks (PBs) was used to transform the information on 3D protein backbone conformation as a ID sequence string. A progressive alignment strategy similar to CLUSTALW was adopted for multiple PB sequence alignment (mulPBA). Highly similar stretches identified by the pairwise alignments are given higher weights during the alignment. The residue equivalences from PB based alignments are used to obtain a three dimensional fit of the structures followed by an iterative refinement of the structural superposition. Systematic comparisons using benchmark datasets of MSTAs underlines that the alignment quality is better than MULTIPROT, MUSTANG and the alignments in HOMSTRAD, in more than 85% of the cases. Comparison with other rigid-body and flexible MSTAs also indicate that mulPBA alignments are superior to most of the rigid-body MSTAs and highly comparable to the flexible alignment methods. (C) 2012 Elsevier Masson SAS. All rights reserved.

Multilayer assemblies of polyelectrolyte-gold nanoparticles for the electrocatalytic oxidation and detection of arsenic(III)

Multilayers of poly(diallyldimethylammonium chloride) (PDDA) and citrate capped Au nanoparticles (AuNPs) anchored on sodium 3-mercapto-1-propanesulfonate modified gold electrode by electrostatic layer-by-layer assembly (LbL) technique are shown to be an excellent architecture for the direct electrochemical oxidation of As(III) species. The growth of successive layers in the proposed LbL architecture is followed by atomic force microscopy, UV-vis spectroscopy, quartz crystal microbalance with energy dissipation, and electrochemistry. The first bilayer is found to show rather different physico-chemical characteristics as compared to the subsequent bilayers, and this is attributed to the difference in the adsorption environments. The analytical utility of the architecture with five bilayers is exploited for arsenic sensing via the direct electrocatalytic oxidation of As(III), and the detection limit is found to be well below the WHO guidelines of 10 ppb. When the non-redox active PDDA is replaced by the redoxactive Os(2,2'-bipyridine)(2)Cl-poly(4-vinylpyridine) polyelectrolyte (PVPOs) in the LbL assembly, the performance is found to be inferior, demonstrating that the redox activity of the polyelectrolyte is futile as far as the direct electro-oxidation of As(III) is concerned. (C) 2012 Elsevier Inc. All rights reserved.

Non-covalent functionalization using lithographically patterned polyelectrolyte multilayers for high-density microarrays

We describe a method to fabricate high-density biological microarrays using lithographic patterning of polyelectrolyte multi layers formed by spin assisted electrostatic layer-by-layer assembly. Proteins or DNA can be immobilized on the polyelectrolyte patterns via electrostatic attachment leading to functional microarrays. As the immobilization is done using electrostatically assembled polyelectrolyte anchor, this process is substrate independent and is fully compatible with a standard semiconductor fabrication process flow. Moreover, the electrostatic assembly of the anchor layer is a fast process with reaction saturation times of the order of a few minutes unlike covalent schemes that typically require hours to reach saturation. The substrate independent nature of this technique is demonstrated by functionalizing glass slides as well as regular transparency sheets using the same procedure. Using a model protein assay, we demonstrate that the non-covalent immobilization scheme described here has competitive performance compared to conventional covalent immobilization schemes described in literature. (C) 2012 Elsevier B.V. All rights reserved.

Sequence and structural basis for chromosomal fragility during translocations in cancer

Chromosomal aberration is considered to be one of the major characteristic features in many cancers. Chromosomal translocation, one type of genomic abnormality, can lead to deregulation of critical genes involved in regulating important physiological functions such as cell proliferation and DNA repair. Although chromosomal translocations were thought to be random events, recent findings suggest that certain regions in the human genome are more susceptible to breakage than others. The possibility of deviation from the usual B-DNA conformation in such fragile regions has been an active area of investigation. This review summarizes the factors that contribute towards the fragility of these regions in the chromosomes, such as DNA sequences and the role of different forms of DNA structures. Proteins responsible for chromosomal fragility, and their mechanism of action are also discussed. The effect of positioning of chromosomes within the nucleus favoring chromosomal translocations and the role of repair mechanisms are also addressed.

Draft Genome Sequence of Staphylococcus aureus ST672, an Emerging Disease Clone from India

We report the draft genome sequence of methicillin-resistant Staphylococcus aureus (MRSA) strain ST672, an emerging disease clone in India, from a septicemia patient. The genome size is about 2.82 Mb with 2,485 open reading frames (ORFs). The staphylococcal cassette chromosome mec (SCCmec) element (type V) and immune evasion cluster appear to be different from those of strain ST772 on preliminary examination.

Generation and analysis of drought stressed subtracted expressed sequence tags from safflower (Carthamus tinctorius L.)

Drought is the most crucial environmental factor that limits productivity of many crop plants. Exploring novel genes and gene combinations is of primary importance in plant drought tolerance research. Stress tolerant genotypes/species are known to express novel stress responsive genes with unique functional significance. Hence, identification and characterization of stress responsive genes from these tolerant species might be a reliable option to engineer the drought tolerance. Safflower has been found to be a relatively drought tolerant crop and thus, it has been the choice of study to characterize the genes expressed under drought stress. In the present study, we have evaluated differential drought tolerance of two cultivars of safflower namely, A1 and Nira using selective physiological marker traits and we have identified cultivar A1 as relatively drought tolerant. To identify the drought responsive genes, we have constructed a stress subtracted cDNA library from cultivar A1 following subtractive hybridization. Analysis of similar to 1,300 cDNA clones resulted in the identification of 667 unique drought responsive ESTs. Protein homology search revealed that 521 (78 %) out of 667 ESTs showed significant similarity to known sequences in the database and majority of them previously identified as drought stress-related genes and were found to be involved in a variety of cellular functions ranging from stress perception to cellular protection. Remaining 146 (22 %) ESTs were not homologous to known sequences in the database and therefore, they were considered to be unique and novel drought responsive genes of safflower. Since safflower is a stress-adapted oil-seed crop this observation has great relevance. In addition, to validate the differential expression of the identified genes, expression profiles of selected clones were analyzed using dot blot (reverse northern), and northern blot analysis. We showed that these clones were differentially expressed under different abiotic stress conditions. The implications of the analyzed genes in abiotic stress tolerance are discussed in our study.

The defect sequence for contractive tuples

We introduce the defect sequence for a contractive tuple of Hilbert space operators and investigate its properties. The defect sequence is a sequence of numbers, called defect dimensions associated with a contractive tuple. We show that there are upper bounds for the defect dimensions. The tuples for which these upper bounds are obtained, are called maximal contractive tuples. The upper bounds are different in the non-commutative and in the commutative case. We show that the creation operators on the full Fock space and the coordinate multipliers on the Drury-Arveson space are maximal. We also study pure tuples and see how the defect dimensions play a role in their irreducibility. (C) 2012 Elsevier Inc. All rights reserved.

Improved Detection of Remote Homologues Using Cascade PSI-BLAST: Influence of Neighbouring Protein Families on Sequence Coverage

Background: Development of sensitive sequence search procedures for the detection of distant relationships between proteins at superfamily/fold level is still a big challenge. The intermediate sequence search approach is the most frequently employed manner of identifying remote homologues effectively. In this study, examination of serine proteases of prolyl oligopeptidase, rhomboid and subtilisin protein families were carried out using plant serine proteases as queries from two genomes including A. thaliana and O. sativa and 13 other families of unrelated folds to identify the distant homologues which could not be obtained using PSI-BLAST. Methodology/Principal Findings: We have proposed to start with multiple queries of classical serine protease members to identify remote homologues in families, using a rigorous approach like Cascade PSI-BLAST. We found that classical sequence based approaches, like PSI-BLAST, showed very low sequence coverage in identifying plant serine proteases. The algorithm was applied on enriched sequence database of homologous domains and we obtained overall average coverage of 88% at family, 77% at superfamily or fold level along with specificity of similar to 100% and Mathew's correlation coefficient of 0.91. Similar approach was also implemented on 13 other protein families representing every structural class in SCOP database. Further investigation with statistical tests, like jackknifing, helped us to better understand the influence of neighbouring protein families. Conclusions/Significance: Our study suggests that employment of multiple queries of a family for the Cascade PSI-BLAST searches is useful for predicting distant relationships effectively even at superfamily level. We have proposed a generalized strategy to cover all the distant members of a particular family using multiple query sequences. Our findings reveal that prior selection of sequences as query and the presence of neighbouring families can be important for covering the search space effectively in minimal computational time. This study also provides an understanding of the `bridging' role of related families.

Enhancing fluorescence signals from aluminium thin films and foils using polyelectrolyte multilayers

In this paper we investigate the application of polyelectrolyte multilayer (PEM) coated metal slides in enhancing fluorescence signal. We observed around eight-fold enhancement in fluorescence for protein incubated on PEM coated on aluminium mirror surface with respect to that of functionalized bare glass slides. The fluorescence intensities were also compared with commercially available FAST (R) slides (Whatman) offering 3D immobilization of proteins and the results were found to be comparable. We also showed that PEM coated on low-cost and commonly available aluminium foils also results in comparable fluorescence enhancement as sputtered aluminium mirrors. Immunoassay was also performed, using model proteins, on aluminium mirror as well as on aluminium foil based devices to confirm the activity of proteins. This work demonstrated the potential of PEMs in the large-scale, roll-to-roll manufacturing of fluorescence enhancements substrates for developing disposable, low-cost devices for fluorescence based diagnostic methods.

The sequence and structure of snake gourd (Trichosanthes anguina) seed lectin, a three-chain nontoxic homologue of type II RIPs

The sequence and structure of snake gourd seed lectin (SGSL), a nontoxic homologue of type II ribosome-inactivating proteins (RIPs), have been determined by mass spectrometry and X-ray crystallography, respectively. As in type II RIPs, the molecule consists of a lectin chain made up of two beta-trefoil domains. The catalytic chain, which is connected through a disulfide bridge to the lectin chain in type II RIPs, is cleaved into two in SGSL. However, the integrity of the three-dimensional structure of the catalytic component of the molecule is preserved. This is the first time that a three-chain RIP or RIP homologue has been observed. A thorough examination of the sequence and structure of the protein and of its interactions with the bound methyl-alpha-galactose indicate that the nontoxicity of SGSL results from a combination of changes in the catalytic and the carbohydrate-binding sites. Detailed analyses of the sequences of type II RIPs of known structure and their homologues with unknown structure provide valuable insights into the evolution of this class of proteins. They also indicate some variability in carbohydrate-binding sites, which appears to contribute to the different levels of toxicity exhibited by lectins from various sources.

Detection of Hepatitis B DNA Sequences on Polyelectrolyte Based Non-Covalently Functionalized Flexible Plastic Substrates

Development of simple functionalization methods to attach biomolecules such as proteins and DNA on inexpensive substrates is important for widespread use of low cost, disposable biosensors. Here, we describe a method based on polyelectrolyte multilayers to attach single stranded DNA molecules to conventional glass slides as well as a completely non-standard substrate, namely flexible plastic transparency sheets. We then use the functionalized transparency sheets to specifically detect single stranded Hepatitis B DNA sequences from samples. We also demonstrate a blocking method for reducing non-specific binding of target DNA sequences using negatively charged polyelectrolyte molecules. The polyelectrolyte based functionalization method, which relies on surface charge as opposed to covalent surface linkages, could be an attractive platform to develop assays on inexpensive substrates for low cost biosensing.

Laser receptive polyelectrolyte thin films doped with biosynthesized silver nanoparticles for antibacterial coatings and drug delivery applications

We report a simple method to fabricate multifunctional polyelectrolyte thin films to load and deliver the therapeutic drugs. The multilayer thin films were assembled by the electrostatic adsorption of poly (allylamine hydrochloride) (PAH) and dextran sulfate (DS). The silver nanoparticles (Ag NPs) biosynthesized from novel Hybanthus enneaspermus leaf extract as the reducing agent were successfully incorporated into the film. The biosynthesized Ag NPs showed excellent antimicrobial activity against the range of enteropathogens, which could be significantly enhanced when used with commercial antibiotics. The assembled silver nano composite multilayer films showed rupture and deformation when they are exposed to laser. The Ag NPs act as an energy absorption center, locally heat up the film and rupture it under laser treatment. The antibacterial drug, moxifloxacin hydrochloride (MH) was successfully loaded into the multilayer films. The total amount of MH release observed was about 63% which increased to 85% when subjected to laser light exposure. Thus, the polyelectrolyte thin film reported in our study has significant potential in the field of remote activated drug delivery, antibacterial coatings and wound dressings. (C) 2013 Elsevier B.V. All rights reserved.

Polyelectrolyte/silver nanocomposite multilayer films as multifunctional thin film platforms for remote activated protein and drug delivery

We demonstrate a nanoparticle loading protocol to develop a transparent, multifunctional polyelectrolyte multilayer film for externally activated drug and protein delivery. The composite film was designed by alternate adsorption of poly(allylamine hydrochloride) (PAH) and dextran sulfate (DS) on a glass substrate followed by nanoparticle synthesis through a polyol reduction method. The films showed a uniform distribution of spherical silver nanoparticles with an average diameter of 50 +/- 20 nm, which increased to 80 +/- 20 nm when the AgNO3 concentration was increased from 25 to 50 mM. The porous and supramolecular structure of the polyelectrolyte multilayer film was used to immobilize ciprofloxacin hydrochloride (CH) and bovine serum albumin (BSA) within the polymeric network of the film. When exposed to external triggers such as ultrasonication and laser light the loaded films were ruptured and released the loaded BSA and CH. The release of CH is faster than that of BSA due to a higher diffusion rate. Circular dichroism measurements confirmed that there was no significant change in the conformation of released BSA in comparison with native BSA. The fabricated films showed significant antibacterial activity against the bacterial pathogen Staphylococcus aureus. Applications envisioned for such drug-loaded films include drug and vaccine delivery through the transdermal route, antimicrobial or anti-inflammatory coatings on implants and drug-releasing coatings for stents. (C) 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Common recognition principles across diverse sequence and structural families of sialic acid binding proteins

Sialic acids form a large family of 9-carbon monosaccharides and are integral components of glycoconjugates. They are known to bind to a wide range of receptors belonging to diverse sequence families and fold classes and are key mediators in a plethora of cellular processes. Thus, it is of great interest to understand the features that give rise to such a recognition capability. Structural analyses using a non-redundant data set of known sialic acid binding proteins was carried out, which included exhaustive binding site comparisons and site alignments using in-house algorithms, followed by clustering and tree computation, which has led to derivation of sialic acid recognition principles. Although the proteins in the data set belong to several sequence and structure families, their binding sites could be grouped into only six types. Structural comparison of the binding sites indicates that all sites contain one or more different combinations of key structural features over a common scaffold. The six binding site types thus serve as structural motifs for recognizing sialic acid. Scanning the motifs against a non-redundant set of binding sites from PDB indicated the motifs to be specific for sialic acid recognition. Knowledge of determinants obtained from this study will be useful for detecting function in unknown proteins. As an example analysis, a genome-wide scan for the motifs in structures of Mycobacterium tuberculosis proteome identified 17 hits that contain combinations of the features, suggesting a possible function of sialic acid binding by these proteins.

Filling-in Void and Sparse Regions in Protein Sequence Space by Protein-Like Artificial Sequences Enables Remarkable Enhancement in Remote Homology Detection Capability

Protein functional annotation relies on the identification of accurate relationships, sequence divergence being a key factor. This is especially evident when distant protein relationships are demonstrated only with three-dimensional structures. To address this challenge, we describe a computational approach to purposefully bridge gaps between related protein families through directed design of protein-like ``linker'' sequences. For this, we represented SCOP domain families, integrated with sequence homologues, as multiple profiles and performed HMM-HMM alignments between related domain families. Where convincing alignments were achieved, we applied a roulette wheel-based method to design 3,611,010 protein-like sequences corresponding to 374 SCOP folds. To analyze their ability to link proteins in homology searches, we used 3024 queries to search two databases, one containing only natural sequences and another one additionally containing designed sequences. Our results showed that augmented database searches showed up to 30% improvement in fold coverage for over 74% of the folds, with 52 folds achieving all theoretically possible connections. Although sequences could not be designed between some families, the availability of designed sequences between other families within the fold established the sequence continuum to demonstrate 373 difficult relationships. Ultimately, as a practical and realistic extension, we demonstrate that such protein-like sequences can be ``plugged-into'' routine and generic sequence database searches to empower not only remote homology detection but also fold recognition. Our richly statistically supported findings show that complementary searches in both databases will increase the effectiveness of sequence-based searches in recognizing all homologues sharing a common fold. (C) 2013 Elsevier Ltd. All rights reserved.