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G Chinea

Publications and source records attributed to G Chinea.

16 recordsLinked to original sources
PubMed2001

Primary structure of two cytolysin isoforms from Stichodactyla helianthus differing in their hemolytic activity.

Sticholysin I (St-I) and sticholysin II (St-II) are cytolysins purified from the sea anemone Stichodactyla helianthus with a high degree of sequence identity (93%) but clearly differenced in their hemolytic activity. In order to go further into the structural determinants for the different behavior of St-I and St-II, we report here the complete amino acid sequences and the consensus secondary structure prediction of both proteins. The complete determination of St-II primary structure confirms the partial revision of cytolysin III amino acid sequence. All nonconservative changes between St-I and St-II are located at the N-terminal. According to our prediction these changes could be located at the same face of an alpha-helix during pore formation events and could account for the observed differences in hemolytic activity between St-I and St-II.

Amino Acid Sequence↗
PubMed2001

Mapping of monoclonal antibodies specific to P64k: a common antigen of several isolates of Neisseria meningitidis.

P64k is a minor outer membrane protein from Neisseria meningitidis. This protein has been produced at high levels in Escherichia coli. We generated a group of monoclonal antibodies (mAbs) against recombinant P64k, which recognise four non-overlapping epitopes, as shown using competition assays with biotinylated mAbs. The P64k sequences involved in mAbs binding were mapped with synthetic overlapping peptides derived from the P64k protein, and located in the previously determined three-dimensional structure of the protein. These antibodies were also characterised by whole-cell ELISA and bactericidal tests against N. meningitidis. Only two of the recognised epitopes were exposed on the bacterial surface, and none of the mAbs showed bactericidal activity. The relationship between these results and the structural data on the epitopes bound by the mAbs is discussed.

Amino Acid Sequence↗
PubMed2000

B-cell epitope mapping of the Neisseria meningitidis P64k protein using overlapping peptides.

A common meningococcal antigen designated P64k has been identified, cloned and expressed in Escherichia coli. The recombinant antigen is highly immunogenic in several animal species and its immunogenicity in healthy human volunteers is under investigation. Recently, P64k has been used as an immunological carrier for weak immunogens. To characterize the B-cell epitopes on P64k, recognized by immune sera obtained from mice, rabbits and monkeys, multiple overlapping peptides were synthesized and screened for antibody binding. Peptides covering the complete sequence of the P64k protein, 59 in all, of 20 amino acids each (overlapped by 10 residues), were synthesized. A number of continuous epitopes were detected with all sera, when immune and pre-immune bleeds were compared. For mouse and monkey sera, a few major antigenic peptides were identified, while the recognition of the rabbit serum was much more heterogeneous. Despite variation in the exact location of continuous epitopes defined by different anti-P64k sera, we found an immunogenic core region within the molecule, composed of amino acids Asp(524)-Gly(533). Consistently, in this protein segment there was an amino acid stretch located in a beta-hairpin loop, which is exposed to the solvent in the previously determined three-dimensional structure of the protein. This region is protruding and accessible to a sphere with a radius of 9 A.

Amino Acid Sequence↗
PubMed2000

Prediction of a common beta-propeller catalytic domain for fructosyltransferases of different origin and substrate specificity.

The three-dimensional (3D) structure of fructan biosynthetic enzymes is still unknown. Here, we have explored folding similarities between reported microbial and plant enzymes that catalyze transfructosylation reactions. A sequence-structure compatibility search using TOPITS, SDP, 3D-PSSM, and SAM-T98 programs identified a beta-propeller fold with scores above the confidence threshold that indicate a structurally conserved catalytic domain in fructosyltransferases (FTFs) of diverse origin and substrate specificity. The predicted fold appeared related to that of neuraminidase and sialidase, of glycoside hydrolase families 33 and 34, respectively. The most reliable structural model was obtained using the crystal structure of neuraminidase (Protein Data Bank file: 5nn9) as template, and it is consistent with the location of previously identified functional residues of bacterial levansucrases (Batista et al., 1999; Song & Jacques, 1999). The sequence-sequence analysis presented here reinforces the recent inclusion of fungal and plant FTFs into glycoside hydrolase family 32, and suggests a modified sequence pattern H-x (2)-[PTV]-x (4)-[LIVMA]-[NSCAYG]-[DE]-P-[NDSC][GA]3 for this family.

Amino Acid Sequence↗
PubMed1999

Mapping of the antigenic regions of streptokinase in humans after streptokinase therapy.

Streptokinase (SK) is efficaciously used as a thrombolytic drug for the treatment of myocardial infarction. Being a bacterial protein, SK is immunogenic in humans. Therefore, resulting from SK therapy, patients become immunized and anti-SK antibody (Ab) titers rise post-treatment. High Ab titers might provoke severe immune reactions during SK therapy and neutralize SK activity, preventing effective thrombolysis. Spot synthesis combined with peptide library techniques is a useful tool for studying protein-peptide interactions on continuous cellulose membranes. Here, we report on the mapping of antigenic regions of SK using a spot-synthesized peptide library and human total sera from patients receiving SK therapy. All tested samples have high anti-SK Ab titers and most of them show significant SK neutralizing capacity. Individual variations in peptide recognition were detected. However, patients treated with SK tend, in general, to show a common regional binding pattern, including residues 1-20, 130-149, 170-189, and 390-399. This is the first study reporting the probing of a cellulose-bound set of peptides with total human sera.

Amino Acid Sequence↗
PubMed1999

Characterization of epitopes on human interleukin-2 using phage displayed-peptide libraries: insights into antibody-peptide interactions.

We have characterized the binding epitopes of two monoclonal antibodies (MAbs) reacting with human Interleukin-2 (IL-2), using a phage display peptide library. The first antibody (CB-IL2.1) recognizes the sequence LSFL, amino acid 72 to amino acid 80, numbered in the IL-2. The second antibody (CB-IL2.2) binds the sequence TTFM (amino acids 101 to 104) located at the opposite site of the four-helix bundle of IL-2. Enzyme-linked immunoadsorbent assay (ELISA) and Western blot using different IL-2 protein construct expressed in bacteria and phage display demonstrate the specificities of this antibody. The data presented here show that the antibodies characterized in this study are raised against linear epitopes and suggest that these epitope are accessible from the outside in the native IL-2 molecule.

Amino Acid Sequence↗
PubMed1998

Structural model for family 32 of glycosyl-hydrolase enzymes.

A structural model is presented for family 32 of the glycosyl-hydrolase enzymes based on the beta-propeller fold. The model is derived from the common prediction of two different threading methods, TOPITS and THREADER. In addition, we used a correlated mutation analysis and prediction of active-site residues to corroborate the proposed model. Physical techniques (circular dichroism and differential scanning calorimetry) confirmed two aspects of the prediction, the proposed all-beta fold and the multi-domain structure. The most reliable three-dimensional model was obtained using the structure of neuraminidase (1nscA) as template. The analysis of the position of the active site residues in this model is compatible with the catalytic mechanism proposed by Reddy and Maley (J. Biol. Chem. 271:13953-13958, 1996), which includes three conserved residues, Asp, Glu, and Cys. Based on this analysis, we propose the participation of one more conserved residue (Asp 162) in the catalytic mechanism. The model will facilitate further studies of the physical and biochemical characteristics of family 32 of the glycosyl-hydrolases.

Amino Acid Sequence↗
PubMed1998

Structural model of Dex protein from Penicillium minioluteum and its implications in the mechanism of catalysis.

The DEX gene encodes an extracellular dextranase (EC 3.2.1.11); this enzyme hydrolyzes the alpha(1,6) glucosidic bond contained in dextran to release small isomaltosaccharides. Sequence analysis has revealed only one homologous sequence, CB-8 protein, from Arthrobacter sp., with 30% sequence identity. The secondary structure prediction for Dex was corroborated by circular dichroism measurements. To explore the possibility that Dex protein might adopt a fold similar to any known structure, we conducted a threading search of a three-dimensional structure database. This search revealed that the Dex sequence is compatible with the galactose oxidase/methanol dehydrogenase/sialidase fold. A structural model of Dex based on these results is physically and biologically plausible and leads to testable predictions, including the prediction that Asp246 and Glu299 might be catalytic residues. Also, according to this model the Dex enzyme has a mechanism of hydrolysis with net inversion of anomeric configuration.

Amino Acid Sequence↗
PubMed1998

Homology modeling, model and software evaluation: three related resources.

MOTIVATION: Homology modeling is rapidly becoming the method of choice for obtaining three-dimensional coordinates for proteins because genome projects produce sequences at a much higher rate than NMR and X-ray laboratories can solve the three-dimensional structures. The quality of protein models will not be immediately clear to novices and support with the evaluation seems to be needed. Expert users are sometimes interested in evaluating the quality of modeling programs rather than the quality of the models themselves. RESULTS: Three servers have been made available to the scientific community: a homology modeling server, a model quality evaluation server and a server that evaluates models built of proteins for which the structure is already known, thereby implicitly evaluating the quality of the modeling program. AVAILABILITY: The modeling-related servers and several structure analysis servers are freely available at http://swift.embl-heidelberg.de/servers/ CONTACT: gert.vriend@embl-heidelberg.de

Computational Biology↗
PubMed1997

Sequence analysis of the structural tbpA gene: protein topology and variable regions within neisserial receptors for transferrin iron acquisition.

The gene coding for the 98-kDa meningococcal outer membrane transferrin binding protein 1 (TbpA) from strain B385 was cloned and sequenced. Sequence comparison among its deduced aminoacid sequence and those from TbpA and the closely related LbpA (lactoferrin binding protein) gene from three different meningococcal strains, and four isolates from two other bacterial pathogens, showed that TbpA variability is confined to five specific segments, designated VR1 (199-287), VR2 (306-381), VR3 (480-546), VR4 (618-651) and VR5 (681-708). The third VR was the most variable among strains both at the nucleotide and amino acid levels. Six additional tbpA genes from different meningococcal strains were cloned and its VR3 sequence determined. On the basis of this data we were able to cluster tbpA genes in two groups: D (bearing a deletion in VR3) and N (nondeleted); all N and D strains belonging to the groups of high or low molecular weight transferrin receptor isotype, respectively. However, by phenogram analysis, the prototypical strain M982 (Group II) was clustered with M990 (B16B6 isotype, Group I). These results point to the existence of important exposed regions as well as to the possibility of horizontal gene exchange involving this locus. A topology model with 14 exposed loops and 28 membrane spanning segments was postulated. According to this tentative analysis, TbpA as well as LbpA proteins should form a gated channel in the neisserial outer membrane. The variable regions were located in the fifth, sixth, eighth, 10th and 11th loops respectively. Among TbpAs VR1, VR2, and VR3 resulted the most relevant regions.

Amino Acid Sequence↗
PubMed1997

Displaying human interleukin-2 on the surface of bacteriophage.

Previous attempts to produce active human Interleukin-2 (hIL-2) in E. coli have failed, due to its aggregation in the form of cytoplasmic inclusion bodies, and the inability of the protein to enter the periplasmic export pathway, when fused to bacterial signal sequences. We have reasoned that these limitations could be overcome by introducing changes in the signal sequence and/or in some hIL-2 residues, not critical for its biological activity; and proceeded to test this hypothesis using a phagemid vector carrying the pelB secretion signal sequence, and the filamentous phage display system. Deletion of the Pro +2 in hIL-2 led to the export of a correct size (processed) molecule to the bacterial periplasm of Su- cells by the phagemid vector. However, this was achieved under growth conditions that would not favor phage assembly in Su+ strains. Changing the hydrophobic core of the leader peptide reversed this situation and allowed phage assembly and display of a pIII/hIL-2 hybrid protein in TG1 cells. The phage-displayed hIL-2 is correctly folded, as judged by its ability to interact with a conformation-specific anti-hIL-2 monoclonal antibody, and maintains its biological activity when tested in a CTLL-2 cell proliferation assay. The changes introduced in hIL-2 and the signal sequence will make possible to use the powerful phage display technology for the selection of high-affinity variants from libraries of hIL-2 mutants.

Amino Acid Sequence↗
PubMed1995

The use of position-specific rotamers in model building by homology.

In this study we concentrate on replacing side chains as a subtask of model building by homology. Two problems arise. How to determine potential low energy rotamers? And how to avoid the combinatorial explosion that results from the combination of many residues for which multiple good rotamers are predicted? We attempt to solve these problems by choosing position-specific rather than generalized rotamers and by sorting the residues that have to be modelled as a function of their freedom in rotamer space. The practical advantages of our method are the quality of the models for cases of high backbone similarity, the small amount of human intervention needed, and the fact that the method automatically estimates the reliability with which each residue has been modeled. Other methods described in this issue are probably more suitable if large backbone rearrangements or loop insertions and deletions need to be modeled.

Bacterial Proteins↗
PubMedDate not supplied

Full window stereo.

Visualisation is the bioinformaticist's most important tool for the study of macromolecules, and being able to see molecules in stereo is a crucial aspect. Stereo vision is based on the principle that each eye is presented with the best possible image of what it would have seen if the object was really there in 3D. The simplest approach to stereo vision is to display the right eye picture on the right half of the screen and the left eye picture on the left half while using a mirror system to ensure that each eye sees what it is supposed to see. More expensive workstations use hardware to alternately display the left and right eye pictures while synchronously blocking the transparency in the right or left lens of the special glasses worn by the user. We present here some simple software that uses inexpensive hardware, originally designed for the computer game industry, to make full screen stereo available on Linux-based PCs. The quality of the stereo vision is similar to the top-of-the-line graphics workstations that are capable of quad-buffering. This stereo option has been incorporated in the XII based version of WHAT IF (Vriend, G. J. Mol. Graphics 1990, 8, 52-56), but the stereo source code is freely available and can easily be incorporated in other visualization packages.

Humans↗