Depth: a web server to compute depth, cavity sizes, detect potential small-molecule ligand-binding cavities and predict the pK(a) of ionizable residues in proteins

Residue depth accurately measures burial and parameterizes local protein environment. Depth is the distance of any atom/residue to the closest bulk water. We consider the non-bulk waters to occupy cavities, whose volumes are determined using a Voronoi procedure. Our estimation of cavity sizes is statistically superior to estimates made by CASTp and VOIDOO, and on par with McVol over a data set of 40 cavities. Our calculated cavity volumes correlated best with the experimentally determined destabilization of 34 mutants from five proteins. Some of the cavities identified are capable of binding small molecule ligands. In this study, we have enhanced our depth-based predictions of binding sites by including evolutionary information. We have demonstrated that on a database (LigASite) of similar to 200 proteins, we perform on par with ConCavity and better than MetaPocket 2.0. Our predictions, while less sensitive, are more specific and precise. Finally, we use depth (and other features) to predict pK(a)s of GLU, ASP, LYS and HIS residues. Our results produce an average error of just <1 pH unit over 60 predictions. Our simple empirical method is statistically on par with two and superior to three other methods while inferior to only one. The DEPTH server (http://mspc.bii.a-star.edu.sg/depth/) is an ideal tool for rapid yet accurate structural analyses of protein structures.

Identification of immuno-dominant antigens of Trypanosoma evansi for detection of chronic trypanosomosis using experimentally infected equines

Trypanosoma evansi is the most extensively distributed trypanosome responsible for disease called surra in livestock in many countries including frequent outbreaks in India. The prevalence of this disease is most commonly reported by standard parasitological detection methods (SPDM); however, antibody ELISA is being in practice by locally produced whole cell lysate (WCL) antigens in many countries. In the present investigation, we attempted to identify and purify immuno dominant, infection specific trypanosome antigens from T. evansi proteome using experimentally infected equine serum by immuno blot. Three immuno dominant clusters of proteins i.e. 62-66 kDa, 52-55 kDa and 41-43 kDa were identified based on their consistent reactivity with donkey sequential serum experimentally infected T. evansi up to 280 days post infection (dpi). The protein cluster of 62-66 kDa was purified in bulk in native form and comparatively evaluated with whole cell lysate antigen (WCL). ELISA and immuno blot showed that polypeptide of this cluster is 100% sensitive in detection of early and chronic infection. Further, this protein cluster was also found immuno reactive against hyper immune serum raised against predominantly 66 kDa exo antigen, revealed that this is a common immunodominant moieties in proteome and secretome of T. evansi.

Gene Transfection in High Serum Levels: Case Studies with New Cholesterol Based Cationic Gemini Lipids

Background: Six new cationic gemini lipids based on cholesterol possessing different positional combinations of hydroxyethyl (-CH2CH2OH) and oligo-oxyethylene -(CH2CH2O)(n)- moieties were synthesized. For comparison the corresponding monomeric lipid was also prepared. Each new cationic lipid was found to form stable, clear suspensions in aqueous media. Methodology/Principal Findings: To understand the nature of the individual lipid aggregates, we have studied the aggregation properties using transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential measurements and X-ray diffraction (XRD). We studied the lipid/DNA complex (lipoplex) formation and the release of the DNA from such lipoplexes using ethidium bromide. These gemini lipids in presence of a helper lipid, 1, 2-dioleoyl phophatidyl ethanol amine (DOPE) showed significant enhancements in the gene transfection compared to several commercially available transfection agents. Cholesterol based gemini having -CH2-CH2-OH groups at the head and one oxyethylene spacer was found to be the most effective lipid, which showed transfection activity even in presence of high serum levels (50%) greater than Effectene, one of the potent commercially available transfecting agents. Most of these geminis protected plasmid DNA remarkably against DNase I in serum, although the degree of stability was found to vary with their structural features. Conclusions/Significance: -OH groups present on the cationic headgroups in combination with oxyethylene linkers on cholesterol based geminis, gave an optimized combination of new genera of gemini lipids possessing high transfection efficiency even in presence of very high percentage of serum. This property makes them preferential transfection reagents for possible in vivo studies.

Fretting wear behaviour of hydroxyapatite-titanium composites in simulated body fluid, supplemented with 5 g l(-1) bovine serum albumin

Damaged articulating joints can be repaired or replaced with synthetic biomaterials, which can release wear debris due to articulation, leading to the osteolysis. In a recent work, it has been shown that it is possible to achieve a better combination of flexural strength/fracture toughness as well as in vitro bioactivity and cytocompatibility properties in spark plasma sintered hydroxyapatite-titanium (HA-Ti) composites. Although hydroxyapatite and titanium are well documented for their good biocompatibility, nanosized hydroxyapatite (HA) and titanium (Ti) particles can cause severe toxicity to cells. In order to address this issue, fretting wear study of HA-Ti composites under dry and wet (1x SBF, supplemented with 5 g l(-1) bovine serum albumin (BSA)) condition was performed to assess the wear resistance as well as wear debris formation, in vitro. The experimental results reveal one order of magnitude lower wear rate for HA-10 wt% Ti (7.5 x 10(-5) mm(3) N-1 m(-1)) composite than monolithic HA (3.9 x 10(-4) mm(3) N-1 m(-1)) in simulated body fluid. The difference in the tribological properties has been analyzed in the light of phase assemblages and mechanical properties. Overall, the results suggest the potential use of HA-Ti composites over existing HA-based biocomposites in orthopedic as well as dental applications.

Understanding the role of domain-domain linkers in the spatial orientation of domains in multi-domain proteins

Inter-domain linkers (IDLs)' bridge flanking domains and support inter-domain communication in multi-domain proteins. Their sequence and conformational preferences enable them to carry out varied functions. They also provide sufficient flexibility to facilitate domain motions and, in conjunction with the interacting interfaces, they also regulate the inter-domain geometry (IDG). In spite of the basic intuitive understanding of the inter-domain orientations with respect to linker conformations and interfaces, we still do not entirely understand the precise relationship among the three. We show that IDG is evolutionarily well conserved and is constrained by the domain-domain interface interactions. The IDLs modulate the interactions by varying their lengths, conformations and local structure, thereby affecting the overall IDG. Results of our analysis provide guidelines in modelling of multi-domain proteins from the tertiary structures of constituent domain components.

Form and Function of Proteins. Historical Background and the Indian Effort in Macromolecular Crystallography

Global efforts in macromolecular crystallography started in the thirties of the last century. However, definitive results began to emerge only in the late fifties and the early sixties. India has a long tradition in crystallography. The country had a head start in theoretical and computational structural biology, thanks to the efforts of G.N. Ramachandran and his colleagues in the fifties and the sixties. However, macromolecular crystallography got off the ground in India only in the eighties, particularly after the Bangalore group received adequate support from the Department of Science and Technology under their Thrust Area Programme. The Bangalore centre was also identified as a national nucleus for the development of the area in the country. Since then work in the area has spread widely and is being carried out by several groups, mainly led by scientists trained at Bangalore or their descendents, in about thirty institutions in India. In addition to the Department of Science and Technology, the effort is now supported by other agencies like the Department of Biotechnology and the Council of Scientific and Industrial Research. The problems addressed by macromolecular crystallographers in India encompass almost all aspects of modern biology. Indian efforts in macromolecular crystallography have also become an important component of the international efforts in the area.

Aptamers as Theranostic Agents: Modifications, Serum Stability and Functionalisation

Aptamers, and the selection process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) used to generate them, were first described more than twenty years ago. Since then, there have been numerous modifications to the selection procedures. This review discusses the use of modified bases as a means of enhancing serum stability and producing effective therapeutic tools, as well as functionalising these nucleic acids to be used as potential diagnostic agents.

Viscoelastic Behavior of Human Lamin A Proteins in the Context of Dilated Cardiomyopathy

Lamins are intermediate filament proteins of type V constituting a nuclear lamina or filamentous meshwork which lines the nucleoplasmic side of the inner nuclear membrane. This protein mesh provides a supporting scaffold for the nuclear envelope and tethers interphase chromosome to the nuclear periphery. Mutations of mainly A-type lamins are found to be causative for at least 11 human diseases collectively termed as laminopathies majority of which are characterised by aberrant nuclei with altered structural rigidity, deformability and poor mechanotransduction behaviour. But the investigation of viscoelastic behavior of lamin A continues to elude the field. In order to address this problem, we hereby present the very first report on viscoelastic properties of wild type human lamin A and some of its mutants linked with Dilated cardiomyopathy (DCM) using quantitative rheological measurements. We observed a dramatic strain-softening effect on lamin A network as an outcome of the strain amplitude sweep measurements which could arise from the large compliance of the quasi-cross-links in the network or that of the lamin A rods. In addition, the drastic stiffening of the differential elastic moduli on superposition of rotational and oscillatory shear stress reflect the increase in the stiffness of the laterally associated lamin A rods. These findings present a preliminary insight into distinct biomechanical properties of wild type lamin A protein and its mutants which in turn revealed interesting differences.

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.

Flocculation behaviour of hematite-kaolinite suspensions in presence of extracellular bacterial proteins and polysaccharides

Cells of Bacillus subtilis exhibited higher affinity towards hematite than to kaolinite. Bacterial cells were grown and adapted in the presence of hematite and kaolinite. Higher amounts of mineral-specific proteinaceous compounds were secreted in the presence of kaolinite while hematite-grown cells produced higher amounts of exopolysaccharides. Extracellular proteins (EP) exhibited higher adsorption density on kaolinite which was rendered more hydrophobic. Hematite surfaces were rendered more hydrophilic due to increased adsorption of extracellular polysaccharides (ECP). Significant surface chemical changes were produced due to interaction between minerals and extracellular proteins and polysaccharides. Iron oxides such as hematite could be effectively removed from kaolinite clays using selective bioflocculation of hematite after interaction with EP and ECP extracted from mineral-grown cells. (C) 2013 Elsevier B.V. All rights reserved.

The relationship between classification of multi-domain proteins using an alignment-free approach and their functions: a case study with immunoglobulins

Establishing functional relationships between multi-domain protein sequences is a non-trivial task. Traditionally, delineating functional assignment and relationships of proteins requires domain assignments as a prerequisite. This process is sensitive to alignment quality and domain definitions. In multi-domain proteins due to multiple reasons, the quality of alignments is poor. We report the correspondence between the classification of proteins represented as full-length gene products and their functions. Our approach differs fundamentally from traditional methods in not performing the classification at the level of domains. Our method is based on an alignment free local matching scores (LMS) computation at the amino-acid sequence level followed by hierarchical clustering. As there are no gold standards for full-length protein sequence classification, we resorted to Gene Ontology and domain-architecture based similarity measures to assess our classification. The final clusters obtained using LMS show high functional and domain architectural similarities. Comparison of the current method with alignment based approaches at both domain and full-length protein showed superiority of the LMS scores. Using this method we have recreated objective relationships among different protein kinase sub-families and also classified immunoglobulin containing proteins where sub-family definitions do not exist currently. This method can be applied to any set of protein sequences and hence will be instrumental in analysis of large numbers of full-length protein sequences.

Correlation between local structural dynamics of proteins inferred from NMR ensembles and evolutionary dynamics of homologues of known structure

Conformational changes in proteins are extremely important for their biochemical functions. Correlation between inherent conformational variations in a protein and conformational differences in its homologues of known structure is still unclear. In this study, we have used a structural alphabet called Protein Blocks (PBs). PBs are used to perform abstraction of protein 3-D structures into a 1-D strings of 16 alphabets (a-p) based on dihedral angles of overlapping pentapeptides. We have analyzed the variations in local conformations in terms of PBs represented in the ensembles of 801 protein structures determined using NMR spectroscopy. In the analysis of concatenated data over all the residues in all the NMR ensembles, we observe that the overall nature of inherent local structural variations in NMR ensembles is similar to the nature of local structural differences in homologous proteins with a high correlation coefficient of .94. High correlation at the alignment positions corresponding to helical and beta-sheet regions is only expected. However, the correlation coefficient by considering only the loop regions is also quite high (.91). Surprisingly, segregated position-wise analysis shows that this high correlation does not hold true to loop regions at the structurally equivalent positions in NMR ensembles and their homologues of known structure. This suggests that the general nature of local structural changes is unique; however most of the local structural variations in loop regions of NMR ensembles do not correlate to their local structural differences at structurally equivalent positions in homologues.

Ramachandran analysis of conserved glycyl residues in homologous proteins of known structure

High conservation of glycyl residues in homologous proteins is fairly frequent. It is commonly understood that glycine tends to be highly conserved either because of its unique Ramachandran angles or to avoid steric clash that would arise with a larger side chain. Using a database of aligned 3D structures of homologous proteins we identified conserved Gly in 288 alignment positions from 85 families. Ninety-six of these alignment positions correspond to conserved Gly residue with (phi, ) values allowed for non-glycyl residues. Reasons for this observation were investigated by in-silico mutation of these glycyl residues to Ala. We found in 94% of the cases a short contact exists between the C atom of the introduced Ala with the atoms which are often distant in the primary structure. This suggests the lack of space even for a short side chain thereby explaining high conservation of glycyl residues even when they adopt (phi, ) values allowed for Ala. In 189 alignment positions, the conserved glycyl residues adopt (phi, ) values which are disallowed for Ala. In-silico mutation of these Gly residues to Ala almost always results in steric hindrance involving C atom of Ala as one would expect by comparing Ramachandran maps for Ala and Gly. Rare occurrence of the disallowed glycyl conformations even in ultrahigh resolution protein structures are accompanied by short contacts in the crystal structures and such disallowed conformations are not conserved in the homologues. These observations raise the doubt on the accuracy of such glycyl conformations in proteins.

PROPENSITIES OF AMINO ACID RESIDUES IN PROTEINS FOR DIFFERENT REGIONS OF THE RAMACHANDRAN MAP

Using a dataset of 1164 crystal structures of largely non-homologous proteins defined at a resolution of 1.5 angstrom or better, we have investigated the (phi,psi) preferences of 20 residue types by considering the residues which occur in loops. Propensities of residue types to occur in the loops with (phi,psi) values in the aa region of the Ramachandran map has a poor correlation coefficient of 0.48 to the Chou-Fasman propensities of the residue types to occur in the a-helical segments. However the correlation coefficient between propensities of residues in loops to adopt beta conformations and those in beta-sheet is much higher (0.95). These observations suggest that a-helix formation is well influenced by the local amino acid sequence while intrinsic preference of residue types for beta-sheet plays a major role in the formation of beta-sheet. The main chain polar groups of residues in loops, that can affect the (phi,psi) values, can be involved in intra-molecular hydrogen bonding. Therefore we investigated further by considering subset of residues in loops with low (0 to 2) number of intra-molecular hydrogen bonds per residue involving main chain polar atoms. For this subset, the correlation coefficients between propensities for alpha-helix and alpha(R) region and between beta-sheet and beta-region are 0.26 and 0.64 respectively. This reiterates higher intrinsic tendency of beta-region favouring residues to adopt beta-sheet than alpha(R) region favouring residues to adopt alpha-helical structure.