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Biomedical subjects

K Gunasekaran

Publications and source records attributed to K Gunasekaran.

At least 19 recordsLinked to original sources
PubMed2006

The structure of complement C3b provides insights into complement activation and regulation.

The human complement system is an important component of innate immunity. Complement-derived products mediate functions contributing to pathogen killing and elimination. However, inappropriate activation of the system contributes to the pathogenesis of immunological and inflammatory diseases. Complement component 3 (C3) occupies a central position because of the manifold biological activities of its activation fragments, including the major fragment, C3b, which anchors the assembly of convertases effecting C3 and C5 activation. C3 is converted to C3b by proteolysis of its anaphylatoxin domain, by either of two C3 convertases. This activates a stable thioester bond, leading to the covalent attachment of C3b to cell-surface or protein-surface hydroxyl groups through transesterification. The cleavage and activation of C3 exposes binding sites for factors B, H and I, properdin, decay accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46), complement receptor 1 (CR1, CD35) and viral molecules such as vaccinia virus complement-control protein. C3b associates with these molecules in different configurations and forms complexes mediating the activation, amplification and regulation of the complement response. Structures of C3 and C3c, a fragment derived from the proteolysis of C3b, have revealed a domain configuration, including six macroglobulin domains (MG1-MG6; nomenclature follows ref. 5) arranged in a ring, termed the beta-ring. However, because neither C3 nor C3c is active in complement activation and regulation, questions about function can be answered only through direct observations on C3b. Here we present a structure of C3b that reveals a marked loss of secondary structure in the CUB (for 'complement C1r/C1s, Uegf, Bmp1') domain, which together with the resulting translocation of the thioester domain provides a molecular basis for conformational changes accompanying the conversion of C3 to C3b. The total conformational changes make many proposed ligand-binding sites more accessible and create a cavity that shields target peptide bonds from access by factor I. A covalently bound N-acetyl-l-threonine residue demonstrates the geometry of C3b attachment to surface hydroxyl groups.

Animals↗
PubMed2006

Muscoid fly populations in tsunami-devastated villages of southern India.

Several coastal villages of southern India were affected by the 26 December 2004 tsunami, and 10,749 people were killed. Investigation carried out in the affected villages during fourth, fifth, and sixth weeks posttsunami showed that the fly density was in the range of 12-91.8 flies per sweep net. In total, 3,259 flies belonging to eight species, namely, Musca domestica L., Musca vicina Macquart, Musca sorbens Wiedemann, Calliphora erythrocephala Robineau-Desvody, Sarcophaga ruficornis F., Chrysomyia sp. Robineau-Desvody, Chlorops sp., and Fannia sp. Robineau-Desvody, were recorded. M. domestica was the predominant species constituting 78.2% of the total flies collected. Density of flies was the highest in temporary shelters constructed for the victims, followed by centralized kitchens and devastated human settlements. Lack of waste control at centralized kitchens nearer to the shelters might be the reason for the high fly density in relief shelters. Under these circumstances, outbreak of fly-borne diseases is likely to be aggravated. Therefore, it is suggested that the ongoing space spraying be supplemented with effective waste control measures to reduce the high density of flies.

Animals↗
PubMed2005

Comparison of the protein-protein interfaces in the p53-DNA crystal structures: towards elucidation of the biological interface.

p53, the tumor suppressor protein, functions as a dimer of dimers. However, how the tetramer binds to the DNA is still an open question. In the crystal structure, three copies of the p53 monomers (containing chains A, B, and C) were crystallized with the DNA-consensus element. Although the structure provides crucial data on the p53-DNA contacts, the active oligomeric state is unclear because the two dimeric (A-B and B-C) interfaces present in the crystal cannot both exist in the tetramer. Here, we address the question of which of these two dimeric interfaces may be more biologically relevant. We analyze the sequence and structural properties of the p53-p53 dimeric interfaces and carry out extensive molecular dynamics simulations of the crystal structures of the human and mouse p53 dimers. We find that the A-B interface residues are more conserved than those of the B-C. Molecular dynamics simulations show that the A-B interface can provide a stable DNA-binding motif in the dimeric state, unlike B-C. Our results indicate that the interface between chains A-B in the p53-DNA complex constitutes a better candidate for a stable biological interface, whereas the B-C interface is more likely to be due to crystal packing. Thus, they have significant implications toward our understanding of DNA binding by p53 as well as p53-mediated interactions with other proteins.

Amino Acid Motifs↗
PubMed2005

Protein-protein interactions: organization, cooperativity and mapping in a bottom-up Systems Biology approach.

Understanding and ultimately predicting protein associations is immensely important for functional genomics and drug design. Here, we propose that binding sites have preferred organizations. First, the hot spots cluster within densely packed 'hot regions'. Within these regions, they form networks of interactions. Thus, hot spots located within a hot region contribute cooperatively to the stability of the complex. However, the contributions of separate, independent hot regions are additive. Moreover, hot spots are often already pre-organized in the unbound (free) protein states. Describing a binding site through independent local hot regions has implications for binding site definition, design and parametrization for prediction. The compactness and cooperativity emphasize the similarity between binding and folding. This proposition is grounded in computation and experiment. It explains why summation of the interactions may over-estimate the stability of the complex. Furthermore, statistically, charge-charge coupling of the hot spots is disfavored. However, since within the highly packed regions the solvent is screened, the electrostatic contributions are strengthened. Thus, we propose a new description of protein binding sites: a site consists of (one or a few) self-contained cooperative regions. Since the residue hot spots are those conserved by evolution, proteins binding multiple partners at the same sites are expected to use all or some combination of these regions.

Binding Sites↗
PubMed2005

The contribution of the Trp/Met/Phe residues to physical interactions of p53 with cellular proteins.

Dynamic molecular interaction networks underlie biological phenomena. Among the many genes which are involved, p53 plays a central role in networks controlling cellular life and death. It not only operates as a tumor suppressor, but also helps regulate hundreds of genes in response to various types of stress. To accomplish these functions as a guardian of the genome, p53 interacts extensively with both nucleic acids and proteins. This paper examines the physical interfaces of the p53 protein with cellular proteins. Previously, in the analysis of the structures of protein-protein complexes, we have observed that amino acids Trp, Met and Phe are important for protein-protein interactions in general. Here we show that these residues are critical for the many functions of p53. Several clusters of the Trp/Met/Phe residues are involved in the p53 protein-protein interactions. Phe19/Trp23 in the TA1 region extensively binds to the transcriptional factors and the MDM2 protein. Trp53/Phe54 in the TA2 region is crucial for transactivation and DNA replication. Met243 in the core domain interacts with 53BP1, 53BP2 and Rad 51 proteins. Met384/Phe385 in the C-terminal region interacts with the S100B protein and the Bromodomain of the CBP protein. Thus, these residues may assist in elucidating the p53 interactions when structural data are not available.

Biophysics↗
PubMed2005

DDT indoor residual spray, still an effective tool to control Anopheles fluviatilis-transmitted Plasmodium falciparum malaria in India.

This study from two districts of Orissa State which are endemic for Plasmodium falciparum transmitted by Anopheles fluviatilis and A. culicifacies investigated the impact of dichlorodiphenyl trichloroethane (DDT) indoor residual spraying, in view of the ongoing discussion on phasing out DDT in India. Based on their high annual parasite incidence and logistical considerations, 26 villages in Malkangiri and 28 in Koraput district were selected for DDT spraying. For comparison, six and four unsprayed villages were chosen from the same districts. In each district, the prevalence of malaria infection and incidence of malaria fever, indoor resting density and parous rate of the vectors, and their susceptibility to DDT were monitored in six and three villages selected randomly from the sprayed and unsprayed groups respectively. Anopheles fluviatilis was susceptible to DDT while A. culicifacies was resistant. DDT residual spraying with 1 g/m(2), was carried out in October-November 2001. Spraying 74-86% of human dwellings and 100% of cattle sheds brought down the indoor resting density of A. fluviatilis by 93-95%. This was associated with a significant reduction of incidence of malaria fever as well as prevalence of malaria infection from November to February in both districts. The spraying also seemed to impact on vector longevity, and a residual effect of DDT on the sprayed walls was observed up to 10-12 weeks despite re-plastering. Hence DDT spraying can still be an effective tool for controlling fluviatilis-transmitted malaria. Although this species is exophilic, its nocturnal resting behaviour facilitates its contact with the sprayed surfaces. DDT is still useful for residual spraying in India, particularly in areas where the vectors are endophilic and not resistant.

Animals↗
PubMed2004

Is allostery an intrinsic property of all dynamic proteins?

Allostery involves coupling of conformational changes between two widely separated binding sites. The common view holds that allosteric proteins are symmetric oligomers, with each subunit existing in "at least" two conformational states with a different affinity for ligands. Recent observations such as the allosteric behavior of myoglobin, a classical example of a nonallosteric protein, call into question the existing allosteric dogma. Here we argue that all (nonfibrous) proteins are potentially allosteric. Allostery is a consequence of re-distributions of protein conformational ensembles. In a nonallosteric protein, the binding site shape may not show a concerted second-site change and enzyme kinetics may not reflect an allosteric transition. Nevertheless, appropriate ligands, point mutations, or external conditions may facilitate a population shift, leading a presumably nonallosteric protein to behave allosterically. In principle, practically any potential drug binding to the protein surface can alter the conformational redistribution. The question is its effectiveness in the redistribution of the ensemble, affecting the protein binding sites and its function. Here, we review experimental observations validating this view of protein allostery.

Allosteric Regulation↗
PubMed2004

Laboratory and field evaluation of Teknar HP-D, a biolarvicidal formulation of Bacillus thuringiensis ssp. israelensis, against mosquito vectors.

Larvicidal efficacy of Teknar HP-D, an improved biolarvicidal formulation of Bacillus thuringiensis ssp. israelensis (Bti), against Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti was determined in the laboratory, and in field the efficacy of the formulation was tested against Cx. quinquefasciatus breeding in cesspits, unused wells and drains. The toxicity of the formulation to Gambusia affinis (larvivorous fish), Notonecta sp. and Diplonychus indicus (water bugs) was also evaluated in the laboratory. Teknar HP-D was field tested at three recommended dosages, 1, 1.5 and 2l/ha, selecting five habitats for each dosage. Another five habitats were kept untreated as controls. Ae. aegypti showed greatest susceptibility to the Bti toxin in the laboratory. In cesspits, all the three dosages caused >80% reduction of pupal recruitment up to day 6 post-treatment, indicating that a weekly application at the lowest would be necessary for sustained control. The residual activity of the formulation was longer in unused wells, causing >80% reduction of pupal recruitment for 17 days from the day of treatment. In controlling pupal recruitment the three dosages produced equal effect. Application of Teknar HP-D at 1 l/ha once in three weeks is therefore recommended to control Cx. quinquefasciatus in unused wells. However, in drains, >80% reduction of pupal recruitment was observed for only 3 days and hence, application of Teknar HP-D at 2 l/ha that caused significantly higher level of reduction twice in a week at 3-day interval is necessary. At dosages from 0.032 to 3.2 mg/l (ppm), Teknar HP-D was non-toxic to Gambusia fish. The two predatory water bugs, Notonecta sp. and Diplonychus indicus that fed on the surviving larvae of Cx. quinquefasciatus exposed to the sub-lethal doses (LC(50) and LC(80)) of Teknar HP-D were safe with out having any mortality.

Animals↗
PubMed2004

Insights into amyloid structural formation and assembly through computational approaches.

Amyloids are long, insoluble ordered fibers. Due to their insolubility, to date the determination of an amyloid structure with an atomic scale resolution has proven to be a difficult task. Under such circumstances, computational approaches are a preferred option, providing the means to build likely models, test their stabilities and figure out the chemistry of their prevailing interactions. Computational models can be validated by targeted experiments, such as introducing mutations and testing for amyloid formation. Computations further provide vehicles for the comprehension of the mechanisms of amyloid seed formation and oligomer toxicity. Nevertheless, computations face an immense hurdle, the outcome of the time scales involved in amyloid formation and the immense sizes of the systems. In an attempt to overcome these, we adopt a strategy that encompasses (1) bioinformatics studies of native proteins containing beta-sheet structures; (2) simulations of shorter peptides; and finally (3) construction of potential oligomeric models and tests of their stabilities. The results are correlated with experimental data where available. Here, we describe the computational methods in simple terms and present an overview of the results. The systems derive from amyloidogenic, disease-related proteins, including gelsolin, beta2-microglobulin, and peptides derived from the prion, Alzheimer's Abeta, IAPP and human calcitonin. Ultimately, obtaining molecular structures should facilitate efforts to therapy and drug design.

Amyloid↗
PubMed2003

Triggering loops and enzyme function: identification of loops that trigger and modulate movements.

Enzyme function often involves a conformational change. There is a general agreement that loops play a vital role in correctly positioning the catalytically important residues. Nevertheless, predicting the functional loops and most importantly their role in enzyme function remains a difficult task. A major reason for this difficulty is that loops that undergo conformational change are frequently not well conserved in their primary sequence. beta1,4-Galactosyltransferase is one such enzyme. There, the amino acid sequence of a long loop that undergoes a large conformational change upon substrate binding is not well conserved. Our molecular dynamics simulations show that the large conformational change in the long loop is brought about by a second, interacting loop. Interestingly, while the structural change of the second loop is much smaller than that of the long loop, its sequence (particularly glycine residues) is highly conserved. We further examine the generality of the proposition that there are loops that trigger movements but nevertheless show little or no structural changes in crystals. We focus on two other enzymes, enolase and lipase. We chose these enzymes, since they too undergo conformational change upon ligand binding, however, they have different folds and different functions. Through multiple sets of simulations we show that the conformational change of the functional loop(s) is brought about through communication of flexibility by triggering loops that have several glycine residues. We further propose that similar to the conservation of common favorable fold types and structural motifs, evolution has also conserved common "skillful" mechanisms. Mechanisms may be conserved across different folds, sequences and functions, with adaptation to specific enzymatic roles.

Apoenzymes↗
PubMed2003

Beta2-microglobulin amyloidosis: insights from conservation analysis and fibril modelling by protein docking techniques.

Current data suggest that globular domains may form amyloids via different mechanisms. Nevertheless, there are indications that the initiation of the process takes place invariably in the less stable segments of a protein domain. We have studied the sequence and structural conservation of beta(2)-microglobulin that deposits into fibrils in dialysis-related amyloidosis. The dataset includes 51 high-resolution non-redundant structures of the antibody constant domain-like proteins (C1) and 132 related sequences. We describe a set of 30 conserved residues. Among them, 23 are conserved structurally, 16 are conserved sequentially and nine are conserved both sequentially and structurally. Strands A (12-18), G (91-95) and D (45-55) are the less conserved and stable segments of the domain, while strands B (22-28), C (36-41), E (62-70) and F (78-83) are the conserved and stable segments. We find that the conserved residues form a cluster with a network of interactions. The observed pattern of conservation is consistent with experimental data including H/D exchange, urea denaturation and limited proteolysis that suggest that strands A and G do not participate in the amyloid fibril. Additionally, the low conservation of strand D is consistent with the observation that this strand may acquire different conformations as seen in crystal structures of bound and isolated beta(2)-microglobulin. We used a docking technique to suggest a model for a fibril via stacking of beta(2)-microglobulin monomers. Our analysis suggests that the favored monomer building block for fibril elongation is the conformation of the isolated beta(2)-microglobulin, without the beta-bulge on strand D and without strands A and G participating in the fibril beta-sheet structure. This monomer retains all the conserved residues and their network of interactions, increasing the likelihood of its existence in solution. The inter-strand interaction between the two (monomer) building blocks forms a new continuous beta-sheet such that addition of monomers results in a fibril model that has the characteristic cross-beta structure.

Amino Acid Sequence↗
PubMed2003

Conservation and amyloid formation: a study of the gelsolin-like family.

The mechanism through which globular proteins transform into amyloid fibrils is still not understood. Here we analyze the structure and sequence conservation to assess the differential stability of segments from two structurally related protein families: the amyloidogenic gelsolin-like and its structurally related cofilin-like. The two families belong to the actin depolymerizing proteins, with a central beta-sheet stacked between 2 and 4 alpha-helices. Although sequentially remote, the two families share regions of high and low conservation and stability. Our results show a highly conserved hydrophobic and aromatic cluster, located at a central buried beta-hairpin. The geometry of the aromatic residues with respect to each other is strictly conserved, suggesting involvement in strand registering and beta-sheet stabilization. Consistent with experiment, we find a region of weak conservation and stability at one of the exposed beta-strands (strand B in the gelsolin-like family). This region was recently found to be affected by a point mutation-mediated destabilization of the human gelsolin domain 2, which facilitates the first proteolytic event in the formation of the amyloidogenic fragment. Thus, both experimental and computational conservation analyses suggest that this unstable region may constitute a first step in amyloid formation. Our analysis uses a recently developed multiple-structure comparison algorithm in which molecules are aligned simultaneously.

Actin Depolymerizing Factors↗
PubMed2003

Interdependence of backbone flexibility, residue conservation, and enzyme function: a case study on beta1,4-galactosyltransferase-I.

Beta1,4-galactosyltransferase-I (beta4Gal-T1) catalyzes the transfer of a galactose from UDP-galactose to N-acetylglucosamine. A recent crystal structure determination of the substrate-bound enzyme reveals a large conformational change, which creates binding sites for the oligosaccharide and alpha-lactalbumin, when compared to the ligand-free structure. The conformational changes take place in a 21-residue-long loop (I345-H365) and in a smaller loop containing a tryptophan residue (W314) flanked by glycines (Y311-G316; Trp loop). A series of molecular dynamics simulations carried out with an implicit solvent model and with explicit water successfully identify flexibility in the two loops and in another interacting loop. These observations are confirmed by limited proteolysis experiments that reveal an intrinsic flexibility of the long loop. The multiple simulation runs starting with the substrate-free structure show that the long loop moves toward its conformation in the ligand-bound structure; however, it gets stabilized in an intermediate position. The Trp loop moves in the opposite direction to that of the long loop, making contacts with residues in the long loop. Remarkably, when the Trp loop is restrained in its starting conformation, no large conformational change takes place in the long loop, indicating residue communication of flexibility. Sequence and structural analysis of the beta4Gal-T1 family with 37 known sequences reveals that in contrast to the unconserved long loop, which undergoes a much larger conformational change, the Trp loop including the glycines is highly conserved. These observations lead us to propose a new functional mechanism that may be conserved by evolution to perform a variety of functions.

Amino Acid Sequence↗
PubMed2002

Efficacy of a floating sustained release formulation of Bacillus thuringiensis ssp. israelensis in controlling Culex quinquefasciatus larvae in polluted water habitats.

Larvicidal efficacy and residual activity of a floating sustained release formulation of Bacillus thuringiensis ssp. israelensis (Bti) was tested in polluted water habitats against Culex quinquefasciatus, the vector of bancroftian filariasis, breeding in cesspools and cesspits. A total of 25 habitats having 65 m(2) water surface area with C. quinquefasciatus immatures were treated with the formulation at the rate of 15 kg/ha. For comparison, in another area, 18 habitats with a water surface area of 70 m(2) were kept untreated. Evaluation was continued up to 179 days with three rounds of Bti application. The mean number of egg rafts did not differ significantly between treated and untreated habitats during the trial period indicating same level of recruitment. However, abundance of larvae and pupae was significantly lower in Bti treated habitats compared to that in untreated ones. The activity of the formulation, with 2 weeks delay in onset compared to treatment, caused >80% reduction in pupal abundance for a period of 30-34 days. Considering this, it is suggested that monthly application of Bti formulation will considerably reduce the recruitment of pupae of C. quinquefasciatus.

Analysis of Variance↗
PubMed2001

Keeping it in the family: folding studies of related proteins.

Investigators have recently turned to studies of protein families to shed light on the mechanism of protein folding. In small proteins for which detailed analysis has been performed, recent studies show that transition-state structure is generally conserved. The number and structures of populated folding intermediates have been found to vary in homologous families of larger (greater than 100-residue) proteins, reflecting a balance of local and global interactions.

Amino Acid Sequence↗
PubMed2000

Development of Wuchereria bancrofti in Culex quinquefasciatus that survived the exposure of sub-lethal dose of Bacillus sphaericus as larvae.

Development of Wuchereria bancrofti in Culex quinquefasciatus emerged from the larvae that survived the exposure of sub-lethal dose of Bacillus sphaericus was examined in the laboratory. Third instar larvae of Cx. quinquefasciatus were treated with B. sphaericus at a sub-lethal dose of 11.35 microg/250 ml. The female mosquitoes that emerged from the survived larvae were fed on microfilaraemic human blood and parasite development was monitored in the fed mosquitoes. Both treated and untreated mosquitoes could ingest microfilaria (mF) equally as there was no significant difference in mF density between them. But, density of developmental stages of the parasite in treated group was significantly lower. Since, there was no mortality of mosquitoes, the lower density of the developmental stages could be attributed to the loss of parasites in the treated mosquitoes. Consequently, the proportion of mosquitoes with infective larvae (L3) and number of L3 were also significantly lower in treated females. Delay in parasite development was also noticed in treated mosquitoes. The present study indicates that B. sphaericus, when applied at sub-lethal dose kills larvae, and in addition, inhibits development of the filarial parasite and consequently reduces L3 yield in adult mosquitoes that emerged from the survived larvae.

Animals↗