| Author | Title | No. | Presentation day | ||
|---|---|---|---|---|---|
| Aguilera E. N. | An intestinal antimicrobial peptide from the Mongolian gerbil, Meriones unguiculatus | 001 | Monday | Poster | |
| Abstract: Pathogenic bacteria are becoming increasingly resistant to conventional antibiotics. For this reason it is necessary to find new antimicrobial agents. Antimicrobial peptides (AMPs) are effector cationic molecules less than 100 amino acids long, gene encoded and ribosomally synthesized. In mammals, like mouse and human, -defensins have been characterized in the small intestine. We attempt to characterize AMPs from the intestinal epithelium of M. unguiculatus. EM showed that Paneth cells of the Lieberkühn crypts are loaded with granules, probably containing AMPs. We separated the intestinal epithelium witn 30 mM EDTA and the AMPs were extracted with 30% acetic acid and 80% methanol, with ultrasonication. The sample was passed through C18 reverse phase and ionic exchange cartridges. Then the peptides were chromatographed repeatedly on a gel filtration column (Waters I-60). The antimicrobial activity was determined using methicillin-resistant Staphylococcus aureus (MRSA). The symmetric peak containing all the antimicrobial activity was subjected to different modes of one- and two-dimensional polyacrylamide gel electrophoresis (native and SDS, urea-acid, with and without DTT) in order to ascertain purity and MW. The antimicrobial activity was obtained in a fraction of less than 3000 MW. We can conclude that there is an intestinal antimicrobial peptide from the intestinal epithelium of the Mongolian gerbil with excellent bactericidal activity vs MRSA. | |||||
BibTeX:
@Poster{N.001,
author = {Aguilera E. N.},
title = {An intestinal antimicrobial peptide from the Mongolian gerbil, Meriones unguiculatus},
booktitle = {Monday},
year = {001},
number = {1}
}
|
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| Antonio-Perez Aurora | DA-CBD, DsbA-CBD and DsbC-CBD assisted the oxidative refolding of hyaluronidase expressed as inclusion bodies | 002 | Tuesday | Poster | |
| Abstract: One of the main concerns regarding the production of recombinant proteins is related to the solubility and folding status of the expressed protein. In order to develop an assisted oxidative refolding chromatographic system we fused the apical domain of GroEL, DsbA and DsbC to the carbohydrate binding domain CBDCex of Celullomonas fimi. These fusion proteins called DA-CBD, DsbA-CBD and DsbC-CBD, free or immobilized on cellulose, assisted the oxidative refolding of denatured and reduced lysozyme as model protein. In this work we used these folding biocatalysts to assist the refolding of a recombinant hyaluronidase (HAsa) from tarantula (Fam. Theraphosidae) expressed as inclusion bodies (IB). HAsa from IB was solubilized and purified by a gel filtration chromatography under reduced conditions. The folding biocatalysts were purified by hydrophobic interaction or Ni- affinity chromatography. HAsa was refolded by yearlysis or 1:100 dilution in the refolding buffer with or without folding biocatalysts. The recovered activity was determined by a turbidimetric assay and zymograms using hyaluronic acid as substrate. The measured activity of native HAsa from tarantula venom was used as control. To isolate the folded HAsa the fusion proteins and misfolded enzyme were adsorbed to cellulose membranes after refolding. HAsa activity was detected until 32 h in spontaneous refolding while in the refolding assisted by AD-CBD, DsbC-CBD and DsbA-CBD, HAsa activity was detected after only 2 h incubation. DA-CBD, DsbA-CBD and DsbC-CBD assisted the oxidative refolding of recombinant protein expressed as IBs. This work was partially supported by grant from DGAPA-UNAM (IN226906) and CONACYT P49987-Z. | |||||
BibTeX:
@Poster{Aurora002,
author = {Antonio-Perez Aurora},
title = {DA-CBD, DsbA-CBD and DsbC-CBD assisted the oxidative refolding of hyaluronidase expressed as inclusion bodies},
booktitle = {Tuesday},
year = {002}
}
|
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| Aispuro-Hernandez Emmanuel | Invertebrate Thioredoxin Is a Potent Antioxidant | 003 | Monday | Poster | |
| Abstract: Nowadays, the redox systems based on glutathione or thioredoxin (TRX) have been deeply studied in higher organisms. Their role in invertebrates that lack an adaptative immune system makes them even more important. In addition, although it is known that TRX protects against oxidative stress its direct antioxidant capacity has not been evaluated. In this study, we present the molecular cloning, recombinant expression, purification and antioxidant activity of the thioredoxin from the Pacific white shrimp Litopenaeus vannamei. The shrimp TRX cDNA sequence resulted in 764 bp that coded for 105 deduced amino acids. Shrimp TRX is 49% identical to human TRX, and has 4 out of 5 cysteines present in the latter protein. ItÂs important to remark that Cys-62 is susceptible for protein nitrosylation as has been recently shown in human TRX (Weichsel A, Brailey JL, Montfort WR. 2007. Biochemistry 46:1219-27). Shrimp TRX was properly expressed and folded in E. coli, and was in disulfide exchange using insulin as a substrate. To evaluate the antioxidant capacity of TRX in solution we used a modified Trolox equivalent antioxidant capacity (TEAC) method. Antioxidant capacity of TRX was significatively higher than the control proteins (bovine serum albumin or lysozyme). Hence, L. vannamei thioredoxin is a potent antioxidant and further investigation ought to be done in order to identify the complete roles of TRX system in this organism. | |||||
BibTeX:
@Poster{Emmanuel003,
author = {Aispuro-Hernandez Emmanuel},
title = { Invertebrate Thioredoxin Is a Potent Antioxidant},
booktitle = {Monday},
year = {003}
}
|
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| Arreola Rodrigo | Crystallographic structure of Triosephosphate Isomerase (TIM) from the cattle tick Boophilus microplus(BmTIM) at 2.4 A resolution. | 004 | Tuesday | Poster | |
| Abstract: Homodimeric TIM enzymes catalyze the interconversion between dihydroxyacetone-phosphate and D-glyceraldehyde-3-phosphate. TIM is a glycolytic enzyme and due to importance of this metabolic pathway it has been proposed as a potential pharmacological target. Olivares-Illana et al. reported in 2006 that some benzothiazole derivatives induce specific inactivation of TIMs of T. brucei and T. cruzi. X-ray protein structures are widely used in the search of agents that exert specific actions or modify the effectiveness of known compounds. This search is based on binding regions and interaction patterns which can be predicted using computational methods based on X-ray structures without bound ligands. This work reports the crystallographic structure of TIM from the cattle tick B. microplus in a ligand free conformation at 2.4 Å resolution. The crystal belongs to the hexagonal space group P6(5) with unit cell parameters a=53.38, b=53.38 and c=278.78 Å. The R and Rfree values are 0.185 and 0.230 respectively. BmTIM is a homodimer, with each monomer of 249 residues forming an (alfa/beta)8 barrel fold, that has nine Cys per subunit; four Cys (25, 43, 51 and 86) are near the interface region. These Cys residues might be a vulnerable point, since their derivatization may very well cause perturbation of intersubunit contacts. The surface regions close to the Cys residues contain a negative charged region and a surface cavity, which might interact with specific molecules. The data set was collected on Advanced Photon Source (APS) Argonne, Ilinnois, USA. Beamline 5-ID (DND-CAT). | |||||
BibTeX:
@Poster{Rodrigo004,
author = {Arreola Rodrigo},
title = {Crystallographic structure of Triosephosphate Isomerase (TIM) from the cattle tick Boophilus microplus(BmTIM) at 2.4 A resolution.},
booktitle = {Tuesday},
year = {004}
}
|
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| Alfaro G. N. | D-Phe in an isomorph of the Molt Inhibiting Hormone of the Mexican crayfish, Procambarus bouvieri. | 005 | Monday | Poster | |
| Abstract: In crustaceans in general, and in the Mexican crayfish P. bouvieri in particular, many physiological processes (gonadal development, glucose levels in the hemolymph and molt cycle, among others) are regulated by peptide hormones synthesized in the medulla terminalis X-organ and accumulated in the sinus gland. This neuropeptide family has 8 common characteristics: 1) acidic pI, 2) hydrophobicity, 3) molecular masses between 8300 and 8400 Da, 4) 72 amino acid residues, 5) blocked N- and C-termini, 6) six cysteines forming three disulfide bridges, 7) lack of histidine, methionine and tryptophan residues, 8) long tracts of sequence identity. The Molt Inhibiting Hormone (MIH) is present in sinus gland extracts as two isomorphs, MIH-I and MIH-II. MIH-I was determined to be a peptide of 72 amino acid residues and a Mr of 8322 Da. The amino acid sequence of MIH-II was determined by means of enzymatic digestions, reverse-phase HPLC, Edman degradation, mass spectromtry, and thin-layer chromatography in quiral plates (CHIR 12381, Merck). The only difference between the two isomorphs consists in a post-translational modification of an L-Phe in MIH-I to a D-Phe in MIH-II at the third position from the N-terminus. MIH shows striking similarity to the Crustacean Hyperglycemic Hormone (CHH) isomorphs of P. bouvieri (90% identity) and to the MIH from Homarus americanus (79% identity) and from Penaeus vannamei (46% identity). This is the same post-translational modification we have found in the CHH-II isomorph of P. bouvieri, and it is the first time that two neuropeptides are so modified in the same organism. | |||||
BibTeX:
@Poster{N.005,
author = {Alfaro G. N.},
title = { D-Phe in an isomorph of the Molt Inhibiting Hormone of the Mexican crayfish, Procambarus bouvieri.},
booktitle = {Monday},
year = {005}
}
|
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| Benites Eleuterio | CRYSTALLOGRAPHIC STUDIES ON GM23, AVARIANT PROTEIN OF monoTIM: AN EXAMPLE OF DIRECTED EVOLUTION TOWARD TPS. | 006 | Tuesday | Poster | |
| Abstract: Abbreviations: monoTIM: monomeric Triose phosphate IsoMerase TPS: Thiamine Phosphate Synthase GM23 is an active monomeric variant resulting from directed evolution by random mutagenesis on the monoTIM from T. brucei. GM23 has been able to complement the activity of TPS in strains where the native TPS gene has been erased. The goal of this study is to analyze structural changes that GM23 acquired and attempt to explain how the mutations improved the affinity for ligands that naturally bind to TPS. GM23 was crystallized using hanging drops in the presence of 2.2M Li2SO4 and 0.1M MES pH 6.5 at 298 K. So far, two X-ray diffraction data sets have been collected: one native ( 2.7 Ǻ) and one soaked (2.9 Ǻ) in the presence of thiamin monophosphate. The localization of a screw-rotation axis in the axial reflections to l, and a twofold symmetry along the crystallographic a axis, suggested that the crystal symmetry belongs to space-groups P6522 or P6322. Phases were determined by molecular replacement using pdb entry 1tri as starting model, subsequently the structure was refined, in space group P6522, and the structural changes were analyzed and compared against pdb file 2tps (TPS structure). The structural information obtained at this resolution show that the principal structural modifications, are in the dimeric interface (as present in wild-type TIM), and in loops 2, 4 and 6. A disulphide bridge was localized between monomers, supporting the existence of a dimers in solution. In addition, the presence of electronic density in the active site, even in the non-soaked crystals, remembers the thiamin monophosphate shape. |
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BibTeX:
@Poster{Eleuterio006,
author = {Benites Eleuterio},
title = {CRYSTALLOGRAPHIC STUDIES ON GM23, AVARIANT PROTEIN OF monoTIM: AN EXAMPLE OF DIRECTED EVOLUTION TOWARD TPS.},
booktitle = {Tuesday},
year = {006}
}
|
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| Alvarez Maria E | The Polyamine Metabolism in Trichomonas vaginalis is Involved in the Cytotoxicity on the HeLa cells | 007 | Monday | Poster | |
| Abstract: Trichomonas vaginalis is the human protozoan parasite responsible of trichomonosis, the most common sexually transmitted infection worldwide. This parasite has numerous cysteine proteinases (CPs), some of which are directly involved in virulence. A 65 kDa CP named CP65 has been involved in trichomonal cytotoxicity over HeLa cell monolayers. The polyamines, putrescine, spermidine, and spermine are abundant and ubiquitous polycations in eukaryotic cells. In T. vaginalis the polyamine metabolism differs from other eukaryotes. This parasite produces and excretes large amounts of putrescine via an energy-generating arginine dihydrolase. The excreted putrescine is used to drive the uptake of spermine via a putrescine:spermine antiporter into the cell. In this work, the involvement of polyamine metabolism on the regulation of T. vaginalis cytotoxicity meyearted by CP65 was investigated using an ornithine decarboxilase inhibitor, 1-4-yearmino-2-butanone (DAB). By cytotoxicity assays, zymograms, as well as Western blots and indirect immunofluorescence experiments using an anti-CP65 antibody, we showed that DAB-treated trichomonads have lower levels of cytotoxicity than untreated organisms, which corresponded to diminished CP65 proteolytic activity in DAB-treated parasites as compared with control untreated trichomonads. Likewise, reduction on the amount of the CP65 protein band and cp65 mRNA in DAB-treated parasites was also observed. These effects were partially reversed by addition of exogenous putrescine to DAB-treated trichomonads. These data showed a direct effect on the polyamine metabolism in the expression regulation of the cp65 gene, a cysteine proteinase involved in trichomonal cytotoxicity to the host cells. | |||||
BibTeX:
@Poster{E007,
author = {Alvarez Maria E},
title = { The Polyamine Metabolism in Trichomonas vaginalis is Involved in the Cytotoxicity on the HeLa cells},
booktitle = {Monday},
year = {007}
}
|
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| Alvarez Carlos | Model Peptides Mimic Functional Properties of Sticholysins, Pore-forming Toxins from a Sea Anemone. | 009 | Monday | Poster | |
| Abstract: Sticholysin I and II (St I and St II) are cytolysins produced by Stichodactyla helianthus. In spite of their 93 % sequence homology, St II is more hemolytic against human erythrocytes than St I. One difference between both toxins resides in their N-termini, where all the non-conservative substitutions are found. In order to establish possible causes of this difference, four peptides from the N-terminus of StI (SELAGTIIDGASLTFEVLDKVLGELGKVSRK, StI1-31 and StI12-31) and StII (ALAGTIIAGASLTFQVLDKVLEELGKVSRK, StII1-30 and StII11-30) were synthesized and their interaction with different interfaces was examined. St II1-30 exhibited the highest hemolytic activity (HA). The peptides were also able to increase the permeability of large unilamellar vesicules and bind to lipid monolayers of different lipid composition. Most of the peptides were more effective when a negatively charged lipid was present with the exception of StI1-31. These results show the importance of electrostatic effects for membrane binding and permeabilizing activity. The presence of two additional negatively charged residues (E2 and D9) in St I1-31 seems to be related to its lower hemolytic and permeabilizing activity, and, by analogy, to the toxin lower HA. The presence of cholesterol in lipid monolayers, elicited a significant increase in St II1-30 binding, possibly due to the formation of ordered lipidic complexes. Altogether, the results suggest that the N-terminus plays an important role in toxin-membrane interaction and provide an explanation at a molecular level for the differences between both isoforms. Supported by FAPESP, CNPq (Brazil), and Ministry of Science and Education (Cuba). | |||||
BibTeX:
@Poster{Carlos009,
author = {Alvarez Carlos},
title = {Model Peptides Mimic Functional Properties of Sticholysins, Pore-forming Toxins from a Sea Anemone.},
booktitle = {Monday},
year = {009}
}
|
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| Luis Blancas-Mejia | Detection of partialy folded intermediate in the folding pathway of an immunoglobulin light chain | 010 | Tuesday | Poster | |
| Abstract: Light chain amyloidosis (amyloidosis AL) is a pathology associated to aggregation of immunoglobulin variable domain light chain (VL) into high ordinate fibril insoluble aggregates. Eventhough a correlation between thermodynamic stability and fibrils formation was found (1), the molecular mechanism still unclear. Some proposed models assume that native state is the precursor of amyloid (2) since in unfolding studies not found evidence of intermediates, but in some cases the evidence suggest that the accumulation of partial unfolding intermediate is the most important factor for fibril aggregation (3). In this work we characterized kinetic and equilibrium folding of lambda 6 germ line (6aJL2). We carried out experiments of intrinsic fluorescence (IF), gel filtration chromatography and differential scanning calorimetry (DSC) with the purpose of detecting an intermediate. IF unfolding curves carried out pre-incubating samples in urea for 24 hours at 25°C and 31°C shown a single sigmoidal transition, but curves pre-incubating at 34°C and 37°C were biphasic and were adjustable to tree-state model, which implies the existence of an intermediate in equilibrium. Under the same conditions gel filtration experiments carried out at 25°C and 37°C; an intermediate was detected. These same experiments incubating samples for 2 minutes not show evidence of intermediate. Kinetic and DSC experiments in urea presence not show intermediate evidence and follow a two-state unfolding process. These results imply that the formation of this intermediate is significantly slower than unfolding of 6aJL2, it is outside of folding pathway and is a irreversible process. 1. Wall, J. et. al. (1999). Biochemistry 38,14101 2. Stevens, F.J. et. al. (1995). Biochemistry 34, 10697 3. Khurana, R. et. al. (2001). Biochemistry 40, 3525 |
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BibTeX:
@Poster{Blancas-Mejia010,
author = {Luis Blancas-Mejia},
title = {Detection of partialy folded intermediate in the folding pathway of an immunoglobulin light chain},
booktitle = {Tuesday},
year = {010}
}
|
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| Alvarez-Escamilla Lupita A | Inactivation of triosephosphate isomerase from Trypanosoma cruzi by three brevifolin carboxylate derivatives. | 011 | Monday | Poster | |
| Abstract: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. According to the World Health Organization, there are 18 million people in the Americas that are affected by this disease, and about 90 million are at risk of contracting infection. The current treatments of Chagas disease are not satisfactory and thus, there is an urgent need to develop new drugs. In view of its importance in glycolysis, some groups have considered triosephosphate isomerase (TIM) from several parasites as a good target for drug design. TIM is an ubiquitous homodimeric house-keeping enzyme that catalyzes the interconversion between glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. In the search of new inhibitors of TIM from T. cruzi (TcTIM), we found that some compounds extracted from Geranium bellum Rose inactivate it in a specie-specific manner. After spectroscopic characterization these compounds were identified as methylbrevifolin carboxylate (C1), ethylbrevifolin carboxylate (C2) and butylbrevifolin carboxylate (C3). Through concentration curves we found that at 6 µM, 8µM and 14µM of C1, C2 and C3 respectively, the activity of TcTIM was reduced fifty percent. Curves at different protein concentrations suggest that these molecules bind at the dimer interface. Also, with molecular simulation of the binding of C1 at TcTIM interface, we found that residues of both monomers are at distance of hydrogen bond from the compound. Besides, assays with human TIM showed that at a concentration of 1mM of the three compounds there was not inactivation. Therefore, these compounds are very selective with respect to the parasite enzyme. | |||||
BibTeX:
@Poster{A011,
author = {Alvarez-Escamilla Lupita A},
title = {Inactivation of triosephosphate isomerase from Trypanosoma cruzi by three brevifolin carboxylate derivatives.},
booktitle = {Monday},
year = {011}
}
|
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| Camacho Zarco A. | Snapshots of natural enzyme function evolution. | 012 | Tuesday | Poster | |
| Abstract: The evolutionary origin of new enzyme functions has been a controversial topic. It has been postulated that catalytic promiscuity could have a critical role in this process by allowing the evolution of new enzyme functions without drastically affecting the original activity (i.e. the weak trade-off hypothesis). The new generalist gene may duplicate, allowing each of the copies to specialize towards catalyzing specifically one of the activities present in the ancestor. The bifunctional enzyme PriA (phosphoribosyl isomerase A) takes part in both histidine (HisA) and tryptophan (TrpF) biosynthesis in most actinomycetes (whose genome lacks a trpF gene). PriA appears to have evolved from a HisA enzyme by moderately compromising its original HisA activity (i.e. weak trade-off) to acquire its TrpF activity. However, the presence of a trpF gene (obtained by lateral gene transfer) in certain Corynebacterium species (a member of actinomyces) prompted us to hypothesized that the HisA/PriA homologues from these organisms (termed neoHisAs) had lost its TrpF activity and regain its HisA proficiency, potentially bringing about evidence in support of the weak trade-off hypothesis. In order to test this possibility, we have cloned, over-expressed and purified one of these homologues, namely, the neoHisA from C. diphtheria, and shown that it indeed lacks TrpF activity. Moreover, a HisA enzyme kinetic assay has been established and preliminary results related to the enzymatic proficiency of this neoHisA will be reported. The evolutionary model composed by HisA-PriA-neoHisA represents the first complete natural history of the evolution of enzyme function. | |||||
BibTeX:
@Poster{A.012,
author = {Camacho Zarco A.},
title = {Snapshots of natural enzyme function evolution.},
booktitle = {Tuesday},
year = {012}
}
|
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| Armenta Dagoberto | Obtaining functional information of the tim barrel by means of a statistically coupled positions analysis in the phosphoribosyl anthranilate isomerase family | 013 | Monday | Poster | |
| Abstract: The present work try to obtain a better understanding of the rules contained in the protein sequences that determine the TIM beta-alpha barrel fold. The orthologous sequences of the family model Phosphoribosyl Anthranilate Isomerase (PRAI), were aligned and analyzed with the Statistical coupling analysis (SCA) method. The couplings values for each position of the alignment were established, generating a global pattern of coevolution. The pattern of coevolutionary functional interactions is sparse therefore just a small set of positions mutually coevolves between the majority that are largely decoupled, the strongly coevolving residues are spatially organized into physically connected networks linking near and distant functional sites of great relevance for the folding process and protein activity (Ranganathan et al., 2005). Our analysis of the family shows 54 residues strongly coupled that determine the core structure of the PRAI barrel and other structural determinants that give versatility to the fold. The reliability of our SCA analysis results are strongly supported by the high degree of coupling in the sites that conforms the hydrophobic cores of the barrel as like the oligomerization interface present in PRAI family, these data agree as well with reported antecedents of molecular dynamics, crystallographic and thermodynamics. We are actually extending the analysis of statistical coupling towards other families of beta-alpha barrels related to PRAI, besides to complement our results with other methodologies that are based on the prediction of critical residues for protein structures. | |||||
BibTeX:
@Poster{Dagoberto013,
author = {Armenta Dagoberto},
title = {Obtaining functional information of the tim barrel by means of a statistically coupled positions analysis in the phosphoribosyl anthranilate isomerase family},
booktitle = {Monday},
year = {013}
}
|
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| Camarillo-Cadena Menandro | Determining the thermal denaturation mechanism of B-glucosidase B from Paenibacillus polymyxa | 014 | Tuesday | Poster | |
| Abstract: β-glucosidase B from Paenibacillus polymyxa, βglB, is a monomeric enzyme, with 448 amino acid residues and 51.5 kDa. It belongs to the family 1 of glycoside hydrolases presenting a (β/α)8 barrel structure. The thermal unfolding of the enzyme is irreversible showing evident aggregation after the solution is cooled down. In this work the thermal unfolding mechanism of BglB was determined by monitoring the structural transition through its ellipticity signal at 220 nm. For this purpose, thermal denaturation scannings at different heating rates and enzyme concentrations were done, determining the transition temperatures, Tm. It became evident that the denaturation is kinetically controlled and independent of the enzyme concentration; this last observation suggests that aggregation is a process subsequent to denaturation. Later, denaturation kinetics at different temperatures were made, determining the kinetic constants (kd) by fitting the data sets to simple exponential decays. Thermal denaturation kinetics was also followed by the hydrolytic activity diminution of the enzyme for a chromogenic substrate, the comparison of both CD and activity data showed that the loss of structure and activity occurs simultaneously. On the other hand, partial thermal scannings were made in the spectropolarimeter, interrupting the heating before the end of the transition and rapidly cooling down the enzyme solution. In these conditions a partial recovery of the CD signal was observed. With all these evidences, we propose that the thermal denaturation of BglB follows the Lumry-Eyring mechanism: N <=>D->A. Where N is the native state, D the denatured state and A the irreversibly aggregated state. | |||||
BibTeX:
@Poster{Menandro014,
author = {Camarillo-Cadena Menandro},
title = {Determining the thermal denaturation mechanism of B-glucosidase B from Paenibacillus polymyxa},
booktitle = {Tuesday},
year = {014}
}
|
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| Arvizu-Flores Aldo A. | The role of Lys48 in antifolate binding to E. coli thymidylate synthase: a thermodynamical approach | 015 | Monday | Poster | |
| Abstract: We have studied E. coli thymidylate synthase (TS) as a model for an antifolate drug target to understand the ligand-binding of anticancer drugs in solution. TS catalyze the folate-dependent methylation of deoxyuridine monophosphate (dUMP) to form thymidine monophosphate. K48 is an invariant non-catalytic residue that stabilizes the negative charge of methylene tetrahydrofolate (the enzyme cofactor) by a water-meyearted hydrogen bond. We used tryptophan fluorescence and isothermal titration calorimetry to study ligand binding in wild-type (WT) and K48Q mutant TS. For ligands, we used propargyl-dideaza folate (PDDF) and the antifolate BW1843U89 (U89), which have different modes of binding to the TS active site. During PDDF binding to WT, Trp fluorescence was quenched while emission at 379 nm appeared, suggesting a fluorescence resonance energy transfer (FRET). Titration of K48Q with PDDF produced Trp quenching but no FRET. Similar Trp quenching of WT and mutant TS was observed in the presence of U89, while FRET was not present. The enthalpic and entropic contributions provided by ITC are considerably different for the binding of the antifolates, validating differences in the mode of binding between them. ITC data also indicates that K48Q has lower affinity for both antifolates, consistent with the fluorescence assays. K48Q mutation in E. coli TS impairs the binding of classical folates such as methylene tetrahydrofolate or PDDF, relative to the WT enzyme. The combination of biophysical techniques is useful in correlating the ligand binding in E. coli TS in solution to protein structure determination. | |||||
BibTeX:
@Poster{A.015,
author = {Arvizu-Flores Aldo A.},
title = { The role of Lys48 in antifolate binding to E. coli thymidylate synthase: a thermodynamical approach},
booktitle = {Monday},
year = {015}
}
|
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| Carrillo Normande | Temperature-Induced Unfolding Reaction of Enolase From Saccharomyces cerevisiae | 016 | Tuesday | Poster | |
| Abstract: Yeast Enolase is a dimeric protein with a modified (βα)8 barrel arrangement, an attractive model to study the folding/unfolding process of proteins. This enzyme catalyses the reversible dehydration of D-2-phosphoglycerate to phosphoenolpyruvate in both glycolysis and gluconeogenesis, requiring Mg2+ or Mn2+ for the enzymatic activity. Several reports dicuss many properties of the enzyme, yet the temperature-induced denaturation is understudied; it is not known what thermal-unfolding pathway this enzyme follows. Therefore, the aims of our study are to describe the heat-induced denaturation process of this glycolytic enzyme, to evaluate the effect of the presence of some ions and chemical groups in such process, and to determine the effect of different heating rates, as well as the reversibility of the reaction. Yeast enolase was heated in buffers containing magnesium, chloride, or acetate ions and in phosphate, at constant heating rates. Changes on the secondary structure were monitored by circular dichroism spectroscopy. The results indicate a higher thermal stability in the presence of acetate and Mg2+ ions, since the transitions occur at higher temperatures, and as judged from changes in the apparent free energy (Gapp) associated to the reaction, in comparison with those without the ions, or the phosphate group. It was also observed that at higher heating rates, transitions appear at higher temperatures, indicating kinetic control of the process. Regarding reversibility, under these conditions, the reaction is non-reversible. We propose that there are interactions between enolase and the acetate and Mg2+ ions, which may be helping in maintaining its native conformation. | |||||
BibTeX:
@Poster{Normande016,
author = {Carrillo Normande},
title = {Temperature-Induced Unfolding Reaction of Enolase From Saccharomyces cerevisiae},
booktitle = {Tuesday},
year = {016}
}
|
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| Bustillo-Ruiz Martin I. | White spot syndrome virus chimeric polypeptide thymidine kinase-Thymidilate kinase as a potencial tarjet for antiviral drugs | 017 | Monday | Poster | |
| Abstract: Novel drug targets are found in viruses that target marine invertebrates. The white spot syndrome virus (WSSV) genus Whispovirus (familiy Nimaviridae) is one of the most devastating viral pathogens of cultured shrimp worldwide. Current research is aimed at understanding the molecular mechanisms of infection, and to lead to effective antiviral agents that prevent disease and economical losses in aquaculture. The circular-double stranded-DNA genome of WSSV has been completely sequenced recently and the thymidine kinase-thymidilate kinase gene is present. Therefore, it is an attractive target for nucleoside analogs and acyclic nucleoside phosphonates with antiviral effect. In this work we did molecular modeling and comparative analysis of the sequence of WSSV TK. The WSSV TK-TMK is highly similar with the vaccinia virus thymidine kinase at the aminoterminal domain, while the C-terminal domain is similar to human thymidilate kinase. The linker region is small and this allowed us to perform a theoretical model for the chimeric viral enzyme. Alignments and molecular modeling using crystal structures of TK in complex with nucleoside analogs as templates, allows us to propose TK-TMK as a target for antiviral drugs by molecules that discriminate between the host and the viral enzymes. Recombinant expression of this enzyme will allow testing this hypothesis and study the differential inhibition in vitro. | |||||
BibTeX:
@Poster{I.017,
author = {Bustillo-Ruiz Martin I.},
title = { White spot syndrome virus chimeric polypeptide thymidine kinase-Thymidilate kinase as a potencial tarjet for antiviral drugs},
booktitle = {Monday},
year = {017}
}
|
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| Celaya Correa Ivan F | Thermodynamic characterization of human triosephosphate isomerase refolding | 018 | Tuesday | Poster | |
| Abstract: Nowadays, protein misfolding and the pathological conditions that this provokes has been subject of intensive studies. This may help to understand and find solutions of neurodegenerative diseases. Some of these disorders in humans are related to the misfolding or point mutations in the homodimeric glycolytic enzyme triosephosphate isomerase. In order to obtain more information about this, we made a kinetic and spectroscopic characterization of triosephosphate isomerase (hTPI), in the presence of guanidine hydrochloride (GuHCl) and urea. The results indicated that after a Âstandard equilibrium time the unfolding and refolding of the enzyme was a hysteretic process and that the activity was almost completely recovered. An active monomeric intermeyearte was found in the renaturation pathway, where the equilibrium was reached. This intermeyearte presented spectroscopic characteristics similar to that of the unfolding state. The Gibbs energy changes obtained for folding and association were ∆Gºfold = -9.1 ± 1.2 kJ/mol , ∆Gºassoc = -91.5 ± 7.7 kJ/mol and ∆Gºtot = -113.7 kJ/mol respectively. | |||||
BibTeX:
@Poster{F018,
author = {Celaya Correa Ivan F},
title = {Thermodynamic characterization of human triosephosphate isomerase refolding},
booktitle = {Tuesday},
year = {018}
}
|
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| Cano Maribel | Discoloration of Direct Black 22 using extra cellular enzyme | 019 | Monday | Poster | |
| Abstract: Effluents discharged from textile dyeing are categorized as one of the most difficult to be degraded by biological, chemical or physical technologies. Direct black 22, is a recalcitrant dye, contains azo linkage, it can be toxic and carcinogenic. In this work enzymes from Trametes versicolor ATCC 8273 a white rot fungi, were used to discolored direct black 22 with concentration 500 ppm. It was used a culture kept on plates with complex medium and growth in liquid medium with wheat straw extract 100 g/L and malt extract agar 20 g/L, at 30 °C, in statistic culture. Enzymatic activity, biomass produced, and discoloration were evaluated at 0, 2, 7 and 11 days of growth. Three replicated were done, together with the control. Laccase activity was measured in the liquid medium and in the mycelium, by monitoring the oxidation of 2,2´-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) at 420 nm and pH 5. Biomass was separated from liquid medium and was gravimetric evaluated. Dye discoloration was measured by UV-VIS spectrophotometer. There was a relation between mycelia growth, laccase activity and dye discoloration. Also it was a decreased in the maximal wavelength signal in the spectral profile in the UV region, especially during the 7 and 11 day of growth of mycelia, but with the liquid medium the signal persisted in the UV region. The laccase activity in mycelia was 0.10656 U and 0.03667 U in the liquid medium, and colour removal was about 50 % with mycelia and 37% with liquid medium, on seventh day of growth. | |||||
BibTeX:
@Poster{Maribel019,
author = {Cano Maribel},
title = {Discoloration of Direct Black 22 using extra cellular enzyme},
booktitle = {Monday},
year = {019}
}
|
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| Cervantes Carlos | Roles of acidic residues in the ChrA bacterial chromate transporter | 020 | Tuesday | Poster | |
| Abstract: The ChrA protein from the bacterium Pseudomonas aeruginosa confers resistance to the oxyanion chromate by its efflux from cellÂs cytoplasm (1). ChrA, of 416 amino acid residues, displays a 13-transmembrane segment (TMS) topology (2). It was recently shown that ChrA pertains to the CHR transporter superfamily, which possesses 135 homologous proteins in bacteria, archaea and fungi (3). Several neutral (4) and basic residues have been previously identified by site-directed mutagenesis as essential for the function of ChrA, and they were assigned structural or functional roles. The purpose of this work was to evaluate the role of acidic amino acids in the structure or function of the ChrA protein. Eleven acidic residues, with a charge conservation of 50% or more with respect to the homologs from subfamilies LCHR2 and LCHR5 of the CHR superfamily, were chosen. Site-directed mutagenesis was carried out by the substitution of selected residues for alanine, using the method of PCR and DpnI digestion in Escherichia coli. The pUChrA plasmid, which contains the wild type chrA gene, was used as a template. The mutations were confirmed by sequencing and the altered genes were transferred to P. aeruginosa. Susceptibility to chromate of the mutants was evaluated in nutrient broth cultures after incubation at 37°C for 18 h. The mutants affected in residues Glu60, Glu230 and Asp281 (present in hydrophilic loops of ChrA) maintained the level of chromate resistance as the strain with the wild type chrA gene, showing that they are not important for the function of the protein. In the mutants altered in residues Glu56 and Asp59 (located in periplasmic loop 1), and Glu83, Glu86, Asp162, Asp330, Asp346 and Asp362 (the six acidic residues present in TMSs in ChrA), the resistance to chromate was drastically abolished, showing that these amino acids are essential for ChrA function. These results suggest that periplasmic loop 1 of ChrA participates in an important manner in the process of chromate efflux. Our data also su ggest that the acidic residues present in TMSs are all involved in generating the correct structure of ChrA in the membrane that is required for its functioning as a chromate transporter. 1. Pimentel, B.E. et al. (2002) FEMS Microbiol. Lett. 212:249-254. 2. Jiménez-Mejía, R. et al. (2006) FEMS Microbiol. Lett. 262:178-184. 3. Díaz-Pérez, C. et al., submitted. 4. Aguilera, S. et al. (2004) FEMS Microbiol. Lett. 232:107-112. -Supported by grants from UMSNH (CIC 2.6) and COECYT (CB070237_3). |
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BibTeX:
@Poster{Carlos020,
author = {Cervantes Carlos},
title = {Roles of acidic residues in the ChrA bacterial chromate transporter},
booktitle = {Tuesday},
year = {020}
}
|
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| Contreras-Vergara C. A. | A crustacean mu-class glutathione S-transferase from the shrimp Litopenaeus vannamei: site-directed mutagenesis of invariant tyrosines Y7 and Y116 | 021 | Monday | Poster | |
| Abstract: Glutathione transferases (GST; E. C. 2.5.1.18) are a diverse family of enzymes found ubiquitously in many organisms. They play a central role in the detoxification of endogenous and xenobiotic compounds. These enzymes catalyze the tripeptide glutathione conjugation to the electrophilic center of lipophilic compounds, increasing their solubility and aiding the excretion from the cell.Y6 and Y115 are key amino acids involved in enzyme-substrate interactions required for catalysis in mu-class GST. These residues provide electrophilic assistance and stabilize substrates through their hydroxyl groups. Two site-directed mutants (Y7F and Y116F) were made to study the role of equivalent residues in the active site of the shrimp Litopenaeus vannamei mu-class GST. Both mutants and wild-type enzymes were expressed in E. coli and purified by GSH-affinity chromatography. The mutants and wild type enzymes catalytic steady-state parameters were determined using CDNB as the second substrate. The primary amino acid sequences were modeled based on a crystal structure of a mu-class GST to provide further insights of the changes at the active site. The Y116F mutant had a small increase in kcat and barely changed its kcat/Km for GSH and CDNB compared to wild-type, similarly with Y7F. Molecular modeling showed that the shrimp GST theoretical model has a H108 residue that may contribute to compensate and lead to a less deleterious change, when conserved tyrosine residues are mutated. This work indicates that shrimp GST is a useful model to understand catalysis mechanisms in this critical enzyme. | |||||
BibTeX:
@Poster{A.021,
author = {Contreras-Vergara C. A.},
title = { A crustacean mu-class glutathione S-transferase from the shrimp Litopenaeus vannamei: site-directed mutagenesis of invariant tyrosines Y7 and Y116},
booktitle = {Monday},
year = {021}
}
|
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| Cruces-Angeles M. Eugenia | Effect of mutation of Cys41 on the thermal unfolding and refolding of Triosephosphate isomerase from Saccharomyces cerevisiae | 022 | Tuesday | Poster | |
| Abstract: Triosephosphate isomerase from Saccharomyces cerevisiae (yTIM) is a homodimeric enzyme which has two cysteine residues. One of these (Cys126) is a conserved residue found in more than 188 TIM sequences. Cys126 is located near the C-end of beta strand 5 and forms part of the beta-barrel hydrophobic core. This residue is thought to be essential for maintaining the folding efficiency of yTIM, since its mutation to Ala or Ser largely decreases the second-order constant for refolding, kr (González-Mondragón, et. al., 2004). The other Cys of yTIM (Cys41, located in beta strand 2) is found in only 47 out of 188 TIM sequences. Here we report that mutant C41S of yTIM display catalytic constants nearly identical to those of the wild-type enzyme, which is an expected finding because Cys41 is approx. 9.0 Å away from the active site. In contrast, it was found that mutation C41S decrease kr as much as mutant C126S does. Activation parameters for the refolding reaction, which were determined from the temperature dependence of kr, provide insightful information on the folding mechanism of the TIM dimer, thus complementing previous studies. | |||||
BibTeX:
@Poster{Eugenia022,
author = {Cruces-Angeles M. Eugenia},
title = {Effect of mutation of Cys41 on the thermal unfolding and refolding of Triosephosphate isomerase from Saccharomyces cerevisiae},
booktitle = {Tuesday},
year = {022}
}
|
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| Corzo Gerardo | Structural similarities and functional convergence of arachnid toxins towards voltage gated sodium channels | 023 | Monday | Poster | |
| Abstract: Scorpions and spiders are phenotypically related (have four pairs of walking legs and no wings) and they belong to the class arachnidae, they have evolved structurally different molecules with similar function that recognize similar receptors. Spider and scorpion molecules target voltage-gated sodium channels (Nav) and they bind to the same extracellular receptors at the Nav, but they share low sequence similarities as well as low secondary and three-dimensional structure correspondence. Spiders toxins are shorter than that of scorpion toxins. However, because of similar functions, the surface analysis of the shorter spider toxins could help to decode the residues in a scorpion toxin involve in similar biological activity. Comparison between the insecticidal spider toxin PaluIT2 and that scorpion toxin Bj-rtx had shown the importance of anionic residues in the toxicity but not for Nav receptor recognition, similarly the spider and scorpion mammalian toxins lack functionality when they had lost specific anionic residues. These observations suggested the idea for using biological inactive arachnid toxins, but with the capacity of receptor recognition to be employed as competitive inhibitors of native toxic scorpion toxins as a way to prevent scorpion intoxications. In this way, we have taken one of the highest toxic peptides for mammals, the toxin CssII from Centruroides suffusus suffusus, as model for analyzing the molecular determinants for ion Nav toxicity and affinity. Acknowledgements: Supported by grants CONACYT-Fondos Mixtos MOR-2004-C02-002 to LDP, CONACyT-49773/24968 and Dirección General de Asuntos del Personal Académico (DGAPA-UNAM) IN226006 to GC. | |||||
BibTeX:
@Poster{Gerardo023,
author = {Corzo Gerardo},
title = { Structural similarities and functional convergence of arachnid toxins towards voltage gated sodium channels},
booktitle = {Monday},
year = {023}
}
|
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| Cruz-Morales Pablo | Functional migration between two different folds sharing a common reaction mechanism. | 024 | Tuesday | Poster | |
| Abstract: E1cB-type eliminations within metabolic pathways often take place via abstraction of an acidic proton and subsequent removal of a leaving group from the substrate. Members of the enolase enzyme superfamily, such as the glycolytic enolase, and type II dehydroquinate dehydratases (DHQase) of the shikimate pathway, share this mechanism of reaction, even when they adopt a modified (beta/alpha)8 and Rossmann folds, respectively. Since previous work in other members of the enolase superfamily has unveiled a broad occurrence of natural enzyme promiscuity, a key feature underpinning functional migration across members of this superfamily, we ought to establish DHQase activity in a selected enolase using protein engineering techniques. This evolutionary pathway, i.e. evolution of DHQase activity within an enolase, was also favored because of the possibility of selecting in vivo the newly evolved variants with DHQase activities using an E. coli aroD minus auxotrophic mutant. Furthermore, the enolase used as template for these experiments was selected from the database taking into account the evolutionary histories of the candidate enzymes. Particular attention was paid to the fact that enolases, which in addition to dehydration of 2-phosphogylcerate into phosphoenolpyruvate during glycolysis, play as well several structural and regulatory roles, implying that only paralogous enolases which had been potentially freed from their functional constraints (and become promiscuous?) were considered. Our preliminary results suggest that common reaction mechanisms, independent of the protein fold adopted by enzymes, are the main evolutionary force driving appearance of novel functions. | |||||
BibTeX:
@Poster{Pablo024,
author = {Cruz-Morales Pablo},
title = {Functional migration between two different folds sharing a common reaction mechanism.},
booktitle = {Tuesday},
year = {024}
}
|
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| De Anda-Aguilar Lorena | On the role of Leu3 in the allosteric transition of glucosamine-6-phosphate deaminase from Escherichia coli. | 025 | Monday | Poster | |
| Abstract: Glucosamine-6-phosphate deaminase from Escherichia coli (EC 3.5.99.6) is an allosteric enzyme composed of six identical subunits of 29.7 kDa. This enzyme catalyses the isomerisation and deamination of glucosamine-6-phosphate (GlcN6P) producing fructose 6-phosphate and ammonium ion. It is allosterically activated by N-acetylglucosamine 6-phosphate (GlcNAc6P) and no allosteric inhibitor is known. The vast amount of structural studies around GlcN6P deaminase makes it a good model for the study of generation and propagation of conformational changes associated to the allosteric transition. The crystallographic structure of the wild type enzyme shows that residues Arg172 and Lys208 bind the phospho group of the substrate and the dead-end inhibitor 2-amino-2-deoxy-6-glucitol 6-phosphate. Our group previously reported that the double mutant Arg172Ala:Lys208Glu is inactive in absence of GlcNAc6P, although its activity can be restored through heterotropic activation. The Km value in the activated condition was very close to that for the wild-type enzyme, while its kcat was about 25-fold lower. The kinetic characterization of the triple mutant Leu3Ile:Arg172Ala:Lys208Glu showed that it totally loses its ability to bind the substrate, even in presence of the allosteric activator. This result strongly suggests that residue Leu3 plays critical role in the packing interactions between subunits around the allosteric site and the local conformational changes that produce the quaternary transition. This work was supported by projects CONACYT (41328-Q) and UNAM-PAPIIT (IN225006). | |||||
BibTeX:
@Poster{Lorena025,
author = {De Anda-Aguilar Lorena},
title = { On the role of Leu3 in the allosteric transition of glucosamine-6-phosphate deaminase from Escherichia coli.},
booktitle = {Monday},
year = {025}
}
|
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| Damian Salvador | Differential expression of Amphiphysin II / Bin1 isoforms as adapter proteins in endocytic meyearted for receptor pathway | 026 | Tuesday | Poster | |
| Abstract: AmphiphysinII/Bin1 is a nucleocytosolic protein with characteristics similar to tumoral growth inhibitors. The N-Terminus of the protein corresponds to an ?-helix called BAR with affinity for curved membranes. On the other hand, the C-Terminus domain known as SH3, presents high affinity for Dinamyn. These characteristics support its ability to function as an adapter protein during the endocytosis process. The central region of the protein can be subjected to dynamic alternative splicing, generating a broad range of isoforms that in most cases show unknown specific functions. Since Bin1 also binds to the oncoprotein c-Myc and is able to induce apoptosis by a caspases independent pathway, it is believed this protein is involved in the complex machinery that modulates the endocytic pathway depending on the different stages of the cell cycle. We cloned by RT-PCR four Amphiphysin II/Bin1 isoforms of cell lines PC12 and CHO-SR. Overexpresion of three of these isoforms show an increment in the level of internalization of DiI labeled LDL particles. The fourth isoform that lacks exon 6 originalling described by us, although presents a normal level of DiI-LDL internalization; we expect to find a modification on its binding capacity to c-Myc. The experiments are on course. | |||||
BibTeX:
@Poster{Salvador026,
author = {Damian Salvador},
title = {Differential expression of Amphiphysin II / Bin1 isoforms as adapter proteins in endocytic meyearted for receptor pathway},
booktitle = {Tuesday},
year = {026}
}
|
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| De La Cruz Valentín | Interaction between recombinant proteins COX2A and COX2B of cytochrome c oxidase of Toxoplasma godii | 027 | Monday | Poster | |
| Abstract: Cytochrome c Oxydase subunit II is encoded in the mitochondrial genome of almost all eukaryots. In chlorophycean algae like Chlamydomonas reinhardtii and Polytomella sp., this subunit is nucleus encoded, and it has been fragmented in two different genes, independently encoded by the cox2a and cox2b genes. It is known that cox2a gene encodes COX IIA subunit, corresponding to the N-terminal of a typical COX II, and the cox2b gene encodes the COX IIB protein, equivalent to the C-terminal of the same subunit. We thought that these separate genes get together to form a heterodimeric COX II subunit that results from the noncovalent assembly of the COX IIA and COX IIB polypeptides in cytochrome c oxidase at the inner mitochondrial membrane. Both subunits have extensions in the C- and N- termi respectively, that could be involved in the interaction and stabilization of the COX2A/COX2B heterodimers. (Pérez-Martínez y col., 2001). The fragmented genes cox2a and cox2b have been identified in Plasmodium and Toxoplasma species (Funes y col., 2002), and the N- and C- terminal extensions have also been identified in the fragmented cox2 subunits of these apicomplexan parasites. In this work, we have cloned the cox2a and cox2b genes from Toxoplasma gondii, and overexpressed the corresponding proteins in Escherichia coli. We have purified the corresponding proteins and analyzed the interaction between the two recombinant cox2 subunits. |
|||||
BibTeX:
@Poster{Valentin027,
author = {De La Cruz Valentín},
title = {Interaction between recombinant proteins COX2A and COX2B of cytochrome c oxidase of Toxoplasma godii},
booktitle = {Monday},
year = {027}
}
|
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| del Pozo-Yauner L | Potential implications of somatic hyper-mutation in lambda 6 light chains amiloidogenicity | 028 | Tuesday | Poster | |
| Abstract: Introduction: Light chain-derived (AL) amyloidosis is a fatal disease characterized by the extracellular deposition of light chain-related components as amyloid fibrils. Notably, the products of the variable domain (VL) gene 6a, the single lambda 6 family member, are preferentially associated with AL. Nowadays, it is yet unknown which particular structural features render lambda 6 light chains almost invariable amyloidogenic. In order to gain insight into the molecular origin of this pathological behavior, we studied the nature and pattern of distribution of somatic mutations in lambda 6 light chains. Material and Methods: One hundred and twenty lambda 6 sequences (49 from AL origin) were compiled from the data banks. The DNA and protein sequences were compared with the germline 6a gene. The frequency of mutation was determined for each position. The differences in volume and chemical nature of the substituting residue with respect to the present in the germline were analyzed. Results: As expected, the mutations were preferentially located at the Complementarity Determining Regions (CDRs) of the domain, although a high frequency of change was also observed at the Framework Regions (FRs). For several amino acids, the level of variability strongly depended on the codon used. Interestingly, in near 25% of the sequences, at least one of the five characteristic salt bridges is eliminated by mutations. We identified numerous mutations that modify potentially key structural residues, as Proline, Glycine and non-polar residues forming the folding nucleus. Conclusion: The observed pattern of mutations in lambda 6 sequences seems to be biased toward destabilizing changes. | |||||
BibTeX:
@Poster{Pozo-Yauner028,
author = {del Pozo-Yauner L},
title = {Potential implications of somatic hyper-mutation in lambda 6 light chains amiloidogenicity},
booktitle = {Tuesday},
year = {028}
}
|
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| De la Mora Eugenio | Preliminar titration studies of the active site of laccase exhibit structural changes as a consequence of rayeartion dose. | 029 | Monday | Poster | |
| Abstract: Together with ascorbate oxidase and ceruloplasmin, laccase belong to the blue multicopper oxidases family. Laccase catalyze the oxidization of a wide range of substrates with the concomitant four-electron reduction of molecular oxygen to water. Four copper atoms, classified according to their spectroscopic features, are needed for catalytic activity: one type-1 (T1), one type-2 (T2), and two type-3, with T2 and T3 forming a trinuclear site. T1 copper accepts and transfers the electron from the substrate to the trinuclear site through a CysÂHis pathway. The transfer of four electrons results in the reduction of O2 to two molecules of water. The precise way in which oxygen is reduced remains unclear. Additional studies are needed in order to determine the precise way in which the reduction occurs. We are presenting the crystallographic structure of Coriolopsis gallica laccase at 1.7 Å resolution as well as a preliminary x-ray titration experiment in which clear changes in the electron density, related to the absorbed dose, are observed. The titration experiment gave us new clues about the reduction process. Acknowledgements To Sonia Rojas for technical assitance. This work was funded by grants from the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT), UNAM (IN228106). |
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BibTeX:
@Poster{Mora029,
author = {De la Mora Eugenio},
title = { Preliminar titration studies of the active site of laccase exhibit structural changes as a consequence of rayeartion dose.},
booktitle = {Monday},
year = {029}
}
|
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| Díaz Salazar Alma J | Equilibrium Unfolding of Triosephosphate Isomerase from Schizosaccharomyces pombe | 030 | Tuesday | Poster | |
| Abstract: Triosephosphate isomerase (TIM) is a glycolytic enzyme that catalyzes the interconversion between dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate. All TIMs isolated so far are homodimers with a subunit molecular mass of 26 Kdal. As shown by denaturation/renaturation experiments as well as designed monomeric forms of the enzyme, the dimeric quaternary structure is a necessary requirement for full catalytic activity. High-resolution structures of homologs from various species have the same topological pattern, a one-domain dimeric (/)8 barrel, in common. Nevertheless, their equilibrium folding pathways that have been determined are different. In this work, the reversible unfolding of homodimeric TIM from Schizosaccharomyces pombe (SpTIM) induced by guanidine hydrochloride (GdnHCl) was characterized under equilibrium conditions. Transition was monitored by change of intrinsic fluorescence and catalytic activity. The unfolding reaction is compatible with a two-state model involving the native dimer and unfolded monomers. In the absence of denaturant, the fluorescence emission spectrum of SpTIM shows a λmax at 322 nm. The unfolding of SpTIM produced a monophasic red shift in the spectral center of mass (SCM) of the emission spectra of 21 nm, with a relative increase in the fluorescence intensity at 350 nm of 80 %, compared to native condition. The SpTIM shows a remarkable resistance against GdnHCl, the C1/2 value, calculated for activity data, was 2.7 M. The total Gibbs energy change for transition ( G oND = 83.1 KJ/mol) is large and similar to that for other TIMs. | |||||
BibTeX:
@Poster{J030,
author = {Díaz Salazar Alma J},
title = {Equilibrium Unfolding of Triosephosphate Isomerase from Schizosaccharomyces pombe},
booktitle = {Tuesday},
year = {030}
}
|
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| Diaz-Sanchez Angel G. | Exploring the Structural Determinants of the Coenzyme Specificity of Betaine Aldehyde Dehydrogenase from Pseudomonas aeruginosa | 031 | Monday | Poster | |
| Abstract: Betaine aldehyde dehydrogenase from the pathogen Pseudomonas aeruginosa is one of the few examples known to date of aldehyde dehydrogenases that use NADP+ as coenzyme. In the three-dimensional structure of the holoenzyme with NADP+, four residues interact with 2´-phosphate group of the ribose of the adenosine moiety: K176, which forms a salt bond, and S178 and R210, each of which are H-bonded to the phosphate through a water molecule. E179, a residue which had been thought incompatible with NADP+ binding, is away from the 2Â-phosphate due to its interaction with R40. To explore the importance of these residues in determining specificity for NADP+, we constructed the mutants K176E, K176A, S178D, S178A, E179P, R40P, and R210A. When expressed in Escherichia coli cells, K176E, S178D, and R40P formed inclusion bodies, whereas K176A, S178A, E179P, and R210A were active and could be purified to homogeneity. Their steady-state and pre-steady state kinetics suggest that neither the kinetic mechanism nor the rate-limiting step were affected by these mutations. Regarding nucleotide binding, the most remarkable changes were: 1) in E179P the affinity for NADP+ was reduced 10-fold, which toghether with a 5-fold reduced kcat for the NADP+-dependent reaction results in a specificity for NADP+ 80-fold smaller than that of the wild type enzyme; this contrasts with a 4-fold increase in the affinity of this mutant for NADPH. 2) A 6-fold reduced affinity for NAD(P)+ in the R210A mutant, which also exhibited cooperative binding of NADP+ and NAD(P)H. Supported by CONACYT 59654 grant. | |||||
BibTeX:
@Poster{G.031,
author = {Diaz-Sanchez Angel G.},
title = {Exploring the Structural Determinants of the Coenzyme Specificity of Betaine Aldehyde Dehydrogenase from Pseudomonas aeruginosa},
booktitle = {Monday},
year = {031}
}
|
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| Diaz-Vergara Norma | Principal motions of the Triosephosphate isomerase from Tripanosoma cruzi at the time scale of 140 ns, by Molecular Dynamics Simulations | 032 | Tuesday | Poster | |
| Abstract: Functional proteins are stable macromolecular structures that undergo certain types of internal motions to carry out their corresponding biological function. Triosephosphate isomerase (TIM) is an ubiquitous homodimeric enzyme with approximately 250 residues per subunit that fold in the so called alpha/beta barrel motif. Its function is to catalyze the interconversion of glyceraldehyde and dihydroxyacetone phosphate in the glycolytic pathway. In this work a molecular dynamics study of the Triosephosphate Isomerase from Trypanosome cruzi (TcTIM), the parasite causing the Chagasdisease, is presented. The backbone-atoms covariance matrix, extracted from a 140 ns long trajectory, was yeargonalized to determine the principal components of the TcTIM overall motion. Our results show that the most significant motions are localized in loops 5, 6 and 7 of both subunits, the loop 5 exhibiting the highest fluctuations. The analysis exposed the loop 6 in an open and closed conformation, which have been previously identified by NMR spectroscopy, and reported to be crucial for the catalytic mechanism of TIMs. The interactions between the catalytic residues in the protein were studied in detail as well. Overall, the results provided in the present work are a contribution towards the understanding of the relevant motions and interactions within the protein. Acknowledgments We are grateful to the Direction General de Servicios de Cómputo Académico (DGSCA) of the Universidad Nacional Autónoma de México (UNAM) for computer time and for their excellent services. |
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BibTeX:
@Poster{Norma032,
author = {Diaz-Vergara Norma},
title = {Principal motions of the Triosephosphate isomerase from Tripanosoma cruzi at the time scale of 140 ns, by Molecular Dynamics Simulations},
booktitle = {Tuesday},
year = {032}
}
|
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| Dominguez-Malfavon Lilianha | Design of fluorescent tagged fusion proteins for the study of RNA-Degradosome protein interaction and distribution | 033 | Monday | Poster | |
| Abstract: Introduction. The RNA-degradosome (RNAD) is a multienzyme complex which functions mainly in mRNA degradation, having also possibly an important role in the maturation of certain rRNA precursors. The major components of the complex are Polynucleotide Phosphorylase, the ATP-dependant helicase RhlB, the Endoribonuclease-E and the glycolytic enzyme Enolase. There are also several minor components present in non stoichiometric quantities, such as Poly-phosphate Kinase and the chaperons GroE and DnaK. The RNAD also makes transient interactions with regulatory components (Hfq which directs the targeted turnover of specific transcripts and the proteins RrrA and RrrB, which control the ribonucleolytic activity of RNaseE). It is important to apply new methodologies that allow the study of interactions among proteins and the distribution of the functional complexes in their native environment inside the living cell. Fluorescence technologies and microscopy imaging can be used with this purpose to address important questions related to protein association and distribution. Methods. Design of the construction vectors was made in silico using Vector NTI v6.0. Chosen fluorescent proteins were fused to resident RNA-Degradosome proteins by recombination in vivo with antibiotic resistance selection. Detection of the fusion proteins was made by polyacrylamide gel electrophoresis and fluorescence capability of the fusions was detected by Confocal Microscopy. Results and Conclusions. The fluorescence of the fusion proteins was detected and microscopy imaging was made indicating RNAD assembling and distribution. These fusion proteins should permit the study of the localization, and interaction among the main components of the RNA-degradosome under different physiological conditions in Escherichia coli. | |||||
BibTeX:
@Poster{Lilianha033,
author = {Dominguez-Malfavon Lilianha},
title = { Design of fluorescent tagged fusion proteins for the study of RNA-Degradosome protein interaction and distribution},
booktitle = {Monday},
year = {033}
}
|
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| Espinosa Adriana | Recombination of distant orthologs for the generation of chimeric TIM barrels | 034 | Tuesday | Poster | |
| Abstract: Both in natural evolution and in protein engineering, DNA recombination has been determinant in expanding protein variability. While homologous recombination increases the probability of viability of the chimeric product by decreasing the probability of deleterious mutations inclusion, mulitpool swapping maximizes the protein space exploration, making it possible to generate new folds. The present work concerns a methodological approach to generate protein variability based on the recombination of distant orthologs (SSAP score < 80.0, sequence identity < 20%) obtained from a metagenomic sample with PCR and degenerate primers. The aim of implementing this approach is to minimize the effects of the trade-off resulting from maximizing space exploration and viability of the chimeras. To prove the system, PRAI E. coli (??)3?4 ? (TIM barrel domain) was complemented with fragments of paralogous genes; based on the screening with a folding reporter (CAT), two folded and stable clones were obtained. To test the amplification process of the distant orthologs, four pairs of degenerate primers were design based on motives (MEME) and blocks (BLOCKS) from multiple alignments of orthologous members of four different TIM barrel superfamilies. The primers were tested in E. coli, where they were proved unspecific. With the optimization of the design of degenerate primers and their utilization on metagenomes a very efficient method can be obtained for the production of protein chimeras useful in industry and in the understanding of the relationship between fold and function. CAT, chloramphenicol acetyltransferase; DNA, deoxyribonucleic acid; E. coli, Escherichia coli; PCR, polymerase chain reaction; PRAI, N-(5Â-phosphoribosyl)-anthranilate isomerase. |
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BibTeX:
@Poster{Adriana034,
author = {Espinosa Adriana},
title = {Recombination of distant orthologs for the generation of chimeric TIM barrels},
booktitle = {Tuesday},
year = {034}
}
|
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| Enriqueta Martinez Rojas | Stereoselective oxidation of gamma-decalactol into gamma-decalactone using Rhodococcus sp DSM 44534 as biocatalyst. | 035 | Monday | Poster | |
| Abstract: The production of fine chemicals and pharmaceuticals with a large application as colorants, vitamins nutraceuticals, cosmetics, pesticides, solvents, etc. actually is already well established. Biotransformations are enzyme or whole cell catalyzed conversions. They are an important tool in organic synthesis, especially for the synthesis of chiral molecules, where the reactions catalyzed may be asymmetric syntheses or the resolutions of racemates. Gamma-Decalactone is an important odorant in many fruits and fermented products. It is contributing to peach, apricot, and strawberry flavors. Microbial processes to produce this flavoring have been patented, although the metabolic pathways involved are not yet completely defined. Here we report the use Rhodococcus sp DSM 44534 as biocatalyst to obtain (R)-gamma-decalactone from racemic gamma-decalactol. Rhodococcus sp strain DTB grew in mineral medium containing ethanol as the sole source of energy and carbon. After 5 days the cells were collected and then used to transform a racemic mixture of gamma-decalactol into (R)--decalactone with living cells with 33% ee having a death cells as control. In addition, the lactol oxidizing enzyme was purified and characterized. The enzyme exhibit a high sequence similarity to catalase. The purified enzyme transformed racemic gamma-decalactol into (R)-gamma-decalactone with 88% ee. Rhodococcus sp grown on ethanol as the sole carbon source contained NAD-dependent dehydrogenases active on a wide range of primary (1°) and secondary (2°) alcohols ranging from 1-pentanol to 2-undecanol, further lactols and aldehydes were also oxidized. The reduction of 2-octanone to 2-octanol by NAD-dependent dehydrogenase in presence of NADH was not observed. | |||||
BibTeX:
@Poster{Rojas035,
author = {Enriqueta Martinez Rojas},
title = { Stereoselective oxidation of gamma-decalactol into gamma-decalactone using Rhodococcus sp DSM 44534 as biocatalyst.},
booktitle = {Monday},
year = {035}
}
|
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| Garcia-Orozco Karina | Overexpression of Thymidylate Synthase from White Spot Syndrome Virus, a potential antiviral drug target | 036 | Tuesday | Poster | |
| Abstract: The white spot syndrome virus (WSSV) is a Whispovirus (familiy Nimaviridae) that attacks penaeid crustacea (shrimp) in aquaculture ponds. The genome of WSSV has been completely sequenced and the thymidylate synthase (wssvTS) gene is present. Therefore, it is an attractive target for future antiviral drug development. The TS coding region of the mature polypeptide was amplified from the cDNA of WSSV infected shrimp using a high-fidelity DNA polymerase (Invitrogen) and specific primers containing restriction sites NdeI and BamHI. wssvTS was overexpressed in E. coli BL21SI using the pET11a vector (Novagen). Expression was monitored by SDS-PAGE and enzymatic activity. The TS WSSV cDNA sequence resulted in a 289 amino acids protein. The expressed protein was soluble, as expected. A 30 kDa protein was overexpressed and purified using ionic exchange chromatography. Using this approach we will be able to produce large amounts of protein for structural, kinetic and thermodynamical studies that led antiviral strategies to control this disease. Supported by grant 48991-Z from CONACYT (Consejo Nacional de Ciencia y Tecnología). |
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BibTeX:
@Poster{Karina036,
author = {Garcia-Orozco Karina},
title = {Overexpression of Thymidylate Synthase from White Spot Syndrome Virus, a potential antiviral drug target},
booktitle = {Tuesday},
year = {036}
}
|
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| Escobar Viviana | Statistical Coupling Analysis on class A beta-lactamases | 037 | Monday | Poster | |
| Abstract: Structure and function determinants remain partially understood, despite considerable progress in protein science. Statistical Coupling Analysis (SCA), described by Lockless & Ranganathan (1999), is a sequence-based analysis that has been performed on several protein families and has demonstrated to accurately identify residue networks crucial for structure and function. SCA quantitatively measures the change in amino acid distribution at one position in an MSA given a perturbation at another position as a statistical coupling energy between them (∆∆Gstat). We performed SCA on an alignment of 242 protein sequences of the class A β-lactamase family including only perturbations present in 85 to 130 sequences to obtain a statistical coupling energy matrix. Further 2D hierarchical clustering was used to identify groups of residues showing the greatest co-evolution. The most important cluster identified includes residues that surround the active site and members of well-characterized secondary structures. Additional sequence, structure and experimental mutants analysis is being performed to determine the role of the identified residues on the structure and function in the class A group of β-lactamases that might describe rules for the whole serine-β-lactamase family fold. Abbreviations: (2D) Two-dimensional, (MSA) Multiple Sequence Alignment, (SCA) Statistical Coupling Analysis |
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BibTeX:
@Poster{Viviana037,
author = {Escobar Viviana},
title = {Statistical Coupling Analysis on class A beta-lactamases},
booktitle = {Monday},
year = {037}
}
|
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| Garcia-Torres Itzhel | An important region involved in the different susceptibility to sulfhydryl agents of two triosephosphate isomerases from related parasites. | 038 | Tuesday | Poster | |
| Abstract: Trypanosoma cruzi and T. brucei are two related parasites that cause Chagas disease and Sleeping sickness respectively. Homodimeric triosephosphate isomerases (TIMs) from these parasites (TcTIM and TbTIM) have an identity of 73% in their amino acid sequences and also have remarkably similar X-ray structures. In both TIMs, there is an interface cysteine (Cys14). Sulfhydryl agents like methylmethane thiosulfonate (MMTS) and dithionitrobenzoic acid (DTNB) act on Cys 14 decreasing enzymatic activity by causing structural alterations. Nonetheless, the reactivity of Cys 14 of TcTIM to sulfhydryl agents is 70-fold higher than that of Cys 14 of TbTIM. To explore what causes that difference, we constructed and characterized kinetically eight chimeric proteins with different regions of TcTIM and TbTIM. Exploring the susceptibility of these enzymes to the action of MMTS and DTNB, we found that a chimera that possesses the first half of the sequence of TcTIM (regions 1-4) and the second half of the sequence of TbTIM (regions 5-8) (Chimera 3) showed a similar behavior to TcTIM. However, a chimera with regions 1-3 of TcTIM and regions 4-8 of TbTIM (Chimera 4) showed a pattern of susceptibility that was intermeyearte between those of TcTIM and TbTIM. The main difference between Chimera 3 and Chimera 4 is the presence of residues 88-119 in the sequence (region 4). Chimeras of TbTIM and TcTIM in which only region 4 of the other TIM was substituted, had a similar behavior to Chimera 4. Our results suggest that region 4 plays an important role in the large differences of susceptibility to the sulfhydryl reagents between TcTIM and TbTIM. | |||||
BibTeX:
@Poster{Itzhel038,
author = {Garcia-Torres Itzhel},
title = {An important region involved in the different susceptibility to sulfhydryl agents of two triosephosphate isomerases from related parasites.},
booktitle = {Tuesday},
year = {038}
}
|
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| Estrada Georgina | Recombinant expression of CssII, a four disulfide-bridged scorpion toxin and functional analysis of its C-terminal region | 039 | Monday | Poster | |
| Abstract: Introduction. CssII is a 66 amino acid neurotoxin from the scorpion Centruroides suffusus suffusus that affects voltage-gated sodium channels (Nav) [Martin et al., 1987]. Based on similarities with other scorpion toxins including Cn2 from Centruroides noxius, we have hypothesized that its C-terminus might be involved in molecular recognition and affinity to Nav channels. Several C-terminal mutants of CssII (N66S, N66H, N66R, T64R/N66S) were produced in E. coli for functional analysis. Materials and Methods. The genes of CssII and its C-terminal mutants (N66S, N66H, N66R or T64R/N66S) were cloned into the expression vector, pQE-30 containing a 6His-tag and a Factor Xa cleavage site. Recombinant protein extraction and purification were performed as reported earlier [Estrada et al., 2007,]. Results and Conclusions. After the in vitro folding and enzymatic cleavage, the recombinant CssII (rCssII) peptide showed toxicity comparable with native toxin indicating, indirectly, its correct folding. The final protein yield indicate that the incorporated mutations were not detrimental for critical folding processes. Although mutants N66S, N66H and N66R were toxic to mice, they were less potent than the rCssII, suggesting a decrement in affinity. Furthermore, mutant T64R/N66S was more toxic than rCssII. The toxin Cn2 has arg and ser in those positions, beeing more toxic than CssII. The LD50s of natives Cn2 and CssII are 0.25 μg and 1.3 μg per 20 g mice, respectivelly. Therefore, the double mutation at the C-terminal of CssII seems to increase the affinity for its Nav receptor. Fine electrophysiological experiments are in process to confirm these results. Acknowledgements: Supported in part by grants CONACYT-Fondos Mixtos MOR-2004-C02-002 to LDP, CONACyT-49773/24968 and Dirección General de Asuntos del Personal Académico (DGAPA-UNAM) IN226006 to GC, and DAGAPA-UNAM IN227507 to LDP. Additional funds came from Institute Bioclón S.A. de C.V. | |||||
BibTeX:
@Poster{Georgina039,
author = {Estrada Georgina},
title = { Recombinant expression of CssII, a four disulfide-bridged scorpion toxin and functional analysis of its C-terminal region},
booktitle = {Monday},
year = {039}
}
|
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| Garduno-Juarez Ramon | Artificial Intelligence and the Protein Folding Problem | 040 | Tuesday | Poster | |
| Abstract: How a specific sequence of amino acids determine in a univocal way the correct form of a protein within an unlimited number of possibilities? This question corresponds to the so called PROTEIN FOLDING PROBLEM (PFP), recognized as one of the most challenging problems of modern biology, whose solution is not a pure intellectual exercise due to its biomedical implications. There is enough experimental evidence to suspect that several human illnesses, such as the AlzheimerÂs disease, ParkinsonÂs disease, cataracts, type II yearbetes and others, are due to a change in the 3-dimensional folding pattern of certain proteins that for hereditary reasons, or due to old age, increase their tendency to misfold. We are interested in understanding how a given amino acid sequence can reach the biological active conformation in finite time, since the PFP corresponds to the mathematical problems known as NP-complete. In this quest we have applied several metaheuristic methods such as Tabu Search, Scatter Search and Genetic Algorithms with conformational memories. Very recently we have focused our attention into Swarm Intelligence (SI). SI is a new computational paradigm based in the distributed problems of auto-organization. Its basic principles are similar to those that determine the behavior of natural systems such as ant colonies, fish schooling, bacterial growth and bird flocking. The most interesting algorithms are the Ant Colony Optimization (ACO) and the Particle Swarm Optimization (PSO). At this moment we are developing some ACO and PSO variants that can be applied to study the PFP problem via computer simulations. |
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BibTeX:
@Poster{Ramon040,
author = {Garduno-Juarez Ramon},
title = {Artificial Intelligence and the Protein Folding Problem},
booktitle = {Tuesday},
year = {040}
}
|
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| Gomez Hernandez Jose A | Optimization of eukaryotic protein expression in prokaryotic systems | 042 | Tuesday | Poster | |
| Abstract: Introduction. The expression of eukaryotic proteins in prokaryotic organisms such as Escherichia coli provides a suitable system to express and study functional properties of any protein. Human Polynucleotide Phosphorylase (hPNPase) is a protein of increasing importance due to its involvement in growth arrest of HO-1 melanome cells when the old-35 gene expression is induced by Type I Interferon. The negligible expression of hPNPase in E. coli is due mostly to codon bias problems for the presence of several rare Arginine (CGA, AGA, AGG), Proline (CCC), Isoleucine (ATA) and Leucine (CTA) codons, that are present in tandem as pairs or triplets in the sequence of the mRNA and this is related to ribosome stalling and low and inefficient expression of the cognate protein, as we have observed in total protein extracts. To overcome this problem, we have designed silent mutations to improve hPNPase expression in E. coli. Methods. We changed the rare codons in tandem for high frequency use ones in bacteria using site-directed mutagenesis. We designed primers of 30-50 bases containing convenient silent mutations; afterward we performed a multiple mutagenesis reaction of the plasmid pVJ40 that contains the old-35 hPNPase cistron. Candidates were screened by hPNPase expression detected by 10% PAGE followed of western-blot using anti-hPNPase antibodies. The changes were confirmed by DNA sequencing. Results. The expression of hPNPase in E. coli was detected in the form of a 86 kDa band by Western blotting. Conclusion. Silent mutations aimed to correct codon bias problems in E. coli improved hPNPse expression allowing a better efficiency in the translation of a eukaryotic protein in a prokaryotic system. | |||||
BibTeX:
@Poster{A042,
author = {Gomez Hernandez Jose A},
title = {Optimization of eukaryotic protein expression in prokaryotic systems},
booktitle = {Tuesday},
year = {042}
}
|
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| Ferreira Tatiana Lopes | Spectroscopic Analisys of Single Tryptophan Mutants of the Bothropstoxin-I (PLA2-Lys49). | 043 | Monday | Poster | |
| Abstract: Bothropstoxin-I (BthTx-I) is a homodimeric Lys49-PLA2 present in the venom of the snake Bothrops jararacussu, which lacks hydrolytic activity against phospholipid substrates, yet permeabilizes membranes by a Ca2+ independent mechanism. The interaction of the BthTx-I with model membranes has been studied by intrinsic tryptophan fluorescence emission (ITFE) spectroscopy. Nine separate mutants have been created each with a unique tryptophan residue located at a different position in the interfacial recognition site (IRS) of the protein. The rapid and efficient Ca2+-independent membrane damage against unilamellar liposomes composed of DPPC/DMPA was unaffected by these substitutions. Experiments using time correlated single photon counting (TCSPC) yielded further information about the structure of protein in solution and were correlated with a blue-shifted emission spectra, increased emission intensity, and changes in the kinetics of chemical modification of tryptophan. Together these data suggest that positions 31, 67 and the C-terminal loop residues 117 and 119 are partially inserted into the membrane. The explanation of the Ca2+-independent membrane damaging activity must therefore rely on models that focus on the interaction of the protein with the outer leaflet of the target membrane. Financial Support by CNPq, FAPESP, FAEPA and PRP-USP | |||||
BibTeX:
@Poster{Lopes043,
author = {Ferreira Tatiana Lopes},
title = { Spectroscopic Analisys of Single Tryptophan Mutants of the Bothropstoxin-I (PLA2-Lys49).},
booktitle = {Monday},
year = {043}
}
|
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| Gonzalez-Segura Lilian | Three dimensional structure of orotate phosphoribosyltransferase from Saccharomyces cerevisae | 044 | Tuesday | Poster | |
| Abstract: Orotate phosphoribosyltransferase (OPRTase) catalyzes the Mg2+-dependent condensation of orotic acid with PRPP (5-α-D-phosphorylribose 1-diphosphate) to yield pyrophosphate and the nucleotide OMP which is converted to UMP by OMP decarboxylase. We have determined the structure of three forms of Saccharomyces cerevisae OPRTase, the free enzyme, a ternary complex between enzyme, Mg2+/PRPP and orotate and the binary product complex between enzyme and bound OMP to resolutions between 2.35 Å and 1.74 Å. Large conformational transitions occur between the apo-enzyme structure and those of the substrate and product complexes. Most notably the upper hood domain rotates down on top of the bound ligands of 19.5 degrees in the OMP structure and 24.6 degrees in the orotate/Mg2+-PRPP structure. In addition, the conserved loop comprised of residues 104-116 moves extensively to sequester the substrates from bulk solvent in the ternary complex when compared to either the product complex or the free enzyme. The orotate and Mg2+/PRPP molecules bound in the ternary complex structure are oriented for proper attack of the N1 atom on the C1Â atom of the ribose ring and this orientation of substrates combined with the positioning of the flexible loop provides a clear picture of a catalytically poised reaction complex for Type I phosphoribosyltransferases. In addition, the structural asymmetry of OMP complex combined with the closure of the flexible loop from one subunit into the active site of the opposing subunit in the ternary complex is consistent with the kinetic data that demonstrate induced non-equivalence and cooperativity of OPRTase. | |||||
BibTeX:
@Poster{Lilian044,
author = {Gonzalez-Segura Lilian},
title = {Three dimensional structure of orotate phosphoribosyltransferase from Saccharomyces cerevisae},
booktitle = {Tuesday},
year = {044}
}
|
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| Figueira Ana Carolina M | Orphan nuclear receptor NGFI-B forms dimers with nonclassical interface | 045 | Monday | Poster | |
| Abstract: The orphan receptor nerve growth factor-induced B (NGFI-B) is a member of the nuclear receptorÂs subfamily 4A (Nr4a). NGFI-B was shown to be capable of binding both as a monomer to an extended half-site containing a single AAAGGTCA motif and also as a homodimer to a widely separated everted repeat, as opposed to a large number of nuclear receptors that recognize and bind specific DNA sequences predominantly as homo- and/or heterodimers. To unveil the structural organization of NGFI-B in solution, we determined the quaternary structure of the NGFI-B LBD by a combination of ab initio procedures from small-angle X-ray scattering (SAXS) data and hydrogenÂdeuterium exchange followed by mass spectrometry. Here we report that the protein forms dimers in solution with a radius of gyration of 2.9 nm and maximum dimension of 9.0 nm. We also show that the NGFI-B LBD dimer is V-shaped, with the opening angle significantly larger than that of classical dimerÂs exemplified by estrogen receptor (ER) or retinoid X receptor (RXR). Surprisingly, NGFI-B dimers formation does not occur via the classical nuclear receptor dimerization interface exemplified by ER and RXR, but instead, involves an extended surface area composed of the loop between helices 2 and 3 and C-terminal fraction of the helix 3. Remarkably, the NGFI-B dimer interface is similar to the dimerization interface earlier revealed for glucocorticoid nuclear receptor (GR), which might be relevant to the recognition of cognate DNA response elements by NGFI-B and to antagonism of NGFI-BÂdependent transcription exercised by GR in cells. | |||||
BibTeX:
@Poster{M045,
author = {Figueira Ana Carolina M},
title = { Orphan nuclear receptor NGFI-B forms dimers with nonclassical interface},
booktitle = {Monday},
year = {045}
}
|
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| Guzman Valeria | Energetics and kinetics of folding of dimeric triosephosphate isomerase of Trypanosoma brucei: contribution of cysteine conserved residues | 046 | Tuesday | Poster | |
| Abstract: Triosephosphate isomerase of Trypanosoma brucei (TbTIM) is a dimeric protein of 250 amino acids per subunit. Until this report, appropriate conditions to achieve reversibility upon denaturation of TbTIM had been unknown. TbTIM has three cysteines (126, 39 and 14). In order to study the contribution to stability and association kinetics of conserved residues (126 and 14), we built two double mutants to determine the effect of absence of these two cysteines and a single mutant to dissect the contribution of Cys39. Cys126 is a strictly conserved residue that is located near to catalytic residues and does not participate in catalysis. Cys14 is located in the dimeric interface and is in contact with other subunit residues. Unfolding and refolding transitions of TbTIM and cysteine mutants were monitored using different spectroscopic parameters. Replacement of Cys39 did not show significant destabilization upon thermal denaturalization, as was shown in double mutants. Tryptophane fluoresence showed a three state fold process that include an inactive monomer for refolding transitions for TbTIM and double cysteine mutants. The first transition corresponds to monomer folding and the second one represents monomer association. TbTIM double mutants were destabilized compared to the wild type. Cys126 and Cys14 stabilize monomer association by 13.2 and 7.2 kJ/mol respectively and Cys14 stabilizes monomer folding by 3.1 kJ/mol, the substitutions of this cysteines also diminished association rate constants compared to wild type. Thus, Cys126 a residue that does not make direct contact in TbTIM interface, contributed to association stability and kinetics more than Cys14. | |||||
BibTeX:
@Poster{Valeria046,
author = {Guzman Valeria},
title = {Energetics and kinetics of folding of dimeric triosephosphate isomerase of Trypanosoma brucei: contribution of cysteine conserved residues},
booktitle = {Tuesday},
year = {046}
}
|
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| Fontes Marcos R. M. | Structural studies with native and complexed PrTX-I from Bothrops genus: insights into myotoxic mechanism | 047 | Monday | Poster | |
| Abstract: Phospholipases A2 (PLA2s) are small calciumÂdependent proteins that cause the liberation of fatty acids and lisophospholipids by hydrolysis of membrane phospholipids. These enzymes are the main components of snake venoms from Bothops genus and are largely studied because of the various pharmacological effects they play in envenomation by snake bites. Considering the broad spectrum of pharmacological effects which includes myotoxic, cytotoxic and hypotensive, a deeper study of these toxins can be very important to the search of new drugs. Piratoxin-I (PrTX-I) is a myotoxic PLA2 homologue from the venom of Bothrops pirajai. Alpha-tocopherol (vitamin E) is involved in the regulation of the production of eicosanoids by inhibition of both PLA2 and cyclooxygenase activities and is believed to be a potencial effective inhibitor for this class of proteins. The present work shows the study of two Lys49-PLA2s from snake venoms on their native and complexed forms with α-tocopherol (1.8 Å resolution) by X-ray crystallographic techniques; additionally, comparative structural studies were performed between the structures obtained and others already available in order to better understand the interaction between these proteins and muscle membranes. After these analyses, we pointed out the residues that probably constitute the myotoxic site of PLA2s from Bothrops genus. The knowledge of this site can be very important for the effective inhibition of these toxins leading to the development of more efficient therapies against bothropic envenomation. Supported by FAPESP, FUNDUNESP, LNLS and CNPq. | |||||
BibTeX:
@Poster{M.047,
author = {Fontes Marcos R. M.},
title = { Structural studies with native and complexed PrTX-I from Bothrops genus: insights into myotoxic mechanism},
booktitle = {Monday},
year = {047}
}
|
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| Heras Horacio | pH stability and Low Resolution Structure of Ovorubin, a protease inhibitor from the eggs of Pomacea canaliculata | 048 | Tuesday | Poster | |
| Abstract: Ovorubin is a 300 KDa thermostable protease inhibitor and the major egg protein of the snail Pomacea canaliculata. It is a glyco-lipo-carotenoprotein containing the carotenoid astaxanthin as a prosthetic group. Here we report ovorubin dimensions and global shape and analyze the effect of pH on stability, using small angle X-ray scattering (SAXS), electron microscopy (EM), circular dichroism, fluorescence and absorption spectroscopy. Analysis of the scattering data and estimation of the shape showed that the protein fits into an ellipsoid with an axial length of 69 Å and a radius of 43 Å. The yearmeter obtained by EM (102 Å) agrees with the maximum pair distance obtained from SAXS results. Quaternary structure was only affected at pH values below 4 showing a reduction in its giration radius and loss of tertiary structure. The absorption spectra and CD of ovorubin at pH values between 2 - 12, showed no significant changes in its intensity or shape, indicating that astaxanthin binding was not affected. Taken together these results indicate that low pH values are triggering the disassembling, but not the unfolding of the oligomer. Ovorubin is therefore the carotenoprotein with the highest stability against pH reported for invertebrates. Considering the wide-range pH stability and the protease inhibitor function, it could very well be that ovorubin is actively involved in the chemical defence of embryos by limiting the predatorÂs ability to digest and use essential nutrients from the eggs, thus rendering the ingestion of eggs antinutritive. | |||||
BibTeX:
@Poster{Horacio048,
author = {Heras Horacio},
title = {pH stability and Low Resolution Structure of Ovorubin, a protease inhibitor from the eggs of Pomacea canaliculata},
booktitle = {Tuesday},
year = {048}
}
|
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| Garcia Sergio A | Expression of a novel superoxide dismutase from Deschampsia antarctica in Escherichia coli | 049 | Monday | Poster | |
| Abstract: Superoxide dismutase (SOD) converts the potentially dangerous superoxide radical (O2Â-) into water and hydrogen peroxide. Deschampsia antarctica is a plant that grows in antarctic territory and survives to extreme low temperature and high UV rayeartion, thus is an ideal candidate to obtain SOD. A cDNA library was generated for D. antarctica from which a Cu/Zn SOD gene was obtained and cloned with and without a histidine tag (His) into Escherichia coli BL21-SI, using pET-28a as expression vector. After protein expression, SOD activity was measured in a zymogram using a commercial bovine erythrocyte SOD as positive control and cell extract of the E. coli BL21-SI/pET-28 as negative control. The results showed both D. antarctica SOD and SOD-His were active and comparable to the commercial enzyme, from which we conclude that the his-tag does not interfere in the activity of the enzyme. Details on the expression, purification and enzymatic characterization are discussed as well. | |||||
BibTeX:
@Poster{A049,
author = {Garcia Sergio A},
title = { Expression of a novel superoxide dismutase from Deschampsia antarctica in Escherichia coli},
booktitle = {Monday},
year = {049}
}
|
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| Herlax V | Fatty acids covalently bound to HlyA are necessary for the oligomerization in membranes | 050 | Tuesday | Poster | |
| Abstract: Alpha-Hemolysin is an extracellular protein toxin (107 kDa) secreted by Escherichia coli that acts at the level of plasma membranes of target eukaryotic cells. Posttranslational modification of the protein with fatty acids is required for all known cytotoxic activities which occur at two internal lysine residues (K564 y K690). In a previous work we demonstrated that in soluble toxin, covalently bound fatty acids promotes the exposure of intrinsically disordered regions. In this context these regions may participate in protein-protein interaction and the toxin promotes the formation of proteo-lipidic pores at lytic concentration, then for this reason we studied the interaction of this toxin with phospholipid membrane using artificial planar lipid membranes composed of asolectin. Addition of nanomolar concentrations of toxin resulted in an increase of bilayer conductance at a concentration-dependent fashion, suggesting that several toxin molecules could be involved in the conductive unit. To obtain conclusive information on the formation of the oligomer on red blood cell membranes by Fluorescent Resonance Energy Transfer (FRET), we used different cystein mutants (K564C, K690C, K344C, R369C) derivatized with fluorescent probes ALEXA-488 or ALEXA-546 (fluorescein and rhodamine derivatives). FRET efficiencies of 30% were measured, confirming that the toxin forms an oligomer. These FRET efficiencies correspond to an oligomeric structure because when changing the donor: acceptor relation FRET efficiencies disminished, evidencing clustering but not random distribution. These results show that HlyA oligomerizases and as mutant K690C does not produce FRET, acyl chains are involved in this process, being acylation at this lysine an essential requirement. | |||||
BibTeX:
@Poster{V050,
author = {Herlax V},
title = {Fatty acids covalently bound to HlyA are necessary for the oligomerization in membranes},
booktitle = {Tuesday},
year = {050}
}
|
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| Garcia Gonzalez Alcione | Interaction of globulin11S of Amaranth with a model membrane | 051 | Monday | Poster | |
| Abstract: The amaranth seed have high contain of storage proteins (globulins and albumins) and have a better-balanced amount of essential amino acids than those found in cereals and legumes. Over the last 20 years, isolated from globulin have become indispensable market commodities for increasing overall protein content and/or imparting functionality and texture, as well as enhanced nutritional value in a variety of food products. Assuming that these globulins can aggregate to form a channel that is use by cells for transporting water, which result in the germination of the seed. The objective of this research is to study the interactions of a model biomembrane made of L-DPPC with the protein globulin11s (Amaranthus Hipochondriacus extract) using the Langmuir balance technique. Monolayers of L-DPPC, protein and a mixture of both in water and in buffer subphases were compared. Brewster Angle Microscopy (BAM) and Atomic Force Microscopy (AFM) were used to characterize the morphology of L-DPPC monolayer, protein monolayer and mixtures of protein with L-DPPC films at the air/water interface. We found that the protein mixes well with the L-DPPC monolayer, forming a coexistence of protein-rich and L-DPPC-rich domains. However, both types of domains liquid-like since on compression we observed fingering phenomena at the interface of between these domains. Also images of protein-rich domains confirm the formation of proteins aggregates, mostly trimers and hexamers, with a hole at the center, which indicates the tendency of the protein to easily aggregate forming hole that might have some relevance to water channels formation in vivo for seed germination. | |||||
BibTeX:
@Poster{Alcione051,
author = {Garcia Gonzalez Alcione},
title = { Interaction of globulin11S of Amaranth with a model membrane},
booktitle = {Monday},
year = {051}
}
|
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| Garcia-Gutierrez Ponciano | Study of the inhibition of the SOD of Taenia solium by computational methods | 053 | Monday | Poster | |
| Abstract: Currently, the use of both computer programs for modelling molecular structures with extensive data bases and docking algorithms are successful tools in the development of chemical compounds of interest in medicine. In this context, the development of novel and potent inhibitors of enzymatic activity of proteins by the formation of protein-ligand complexes has deserved great attention. In this work, we present our results on the use of computational methods of docking in order to design, synthesize and test some organic molecules that inhibits the enzymatic activity of the protein superoxide dismutase (SOD-Cu/Zn) of the parasite Taenia solium (T. solium). The data base of compounds with pharmacological potential was LeadQuest, which offers about 2 million structural conformers of approximatively 50 thousand compounds. Also, the interaction of some benzimidazole derivaties on the structure of superoxide dismutase, using the XRD data, was studied. In general, we present and analyze a list of potentially inhibiting molecules of the SOD-Cu/Zn activity, which will be synthesized and evaluated. | |||||
BibTeX:
@Poster{Ponciano053,
author = {Garcia-Gutierrez Ponciano},
title = { Study of the inhibition of the SOD of Taenia solium by computational methods},
booktitle = {Monday},
year = {053}
}
|
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| Hernandez Cuevas Nora A | Generation of EhGEF1 protein mutants from Entamoeba histolytica | 054 | Tuesday | Poster | |
| Abstract: The Rho family GTPases are molecular switches which are involved in the majority of actin-dependent processes such as those related to migration, actin cytoskeleton, morphogenesis, phagocytosis, cell polarity, cell cycle progression and gene expression. The Rho GTPases are low molecular weight proteins which have a cycle in two states: one inactive GDP-bound and the other active GTP-bound. The Rho family GTPases cycle of activation and inactivation is regulated by the GEF proteins or Dbl family guanine nucleotide exchange factors. These proteins have an important role as positive regulators of the Rho proteins. The GEF proteins are able to bind to their inactive target GTPase bound to GDP and to promote GDP disassociation so that the latter can be activated in GTP binding. Recently our study group reported the first GEF in parasites; the EhGEF1 protein from Entamoeba histolytica: this protein is able to promote the guanine nucleotide exchange on the EhRacG and EhRho1 GTPases of amoeba. In this study, we produced models of the EhGEF1-EhRacG and EhGEF1-EhRho1 complexes. Through mutation in silico we estimated the contribution of the residues of the interface region of both complexes. We identified two EhGEF1 residues not preserved amongst the members of the Dbl family, which are important for the selective activity on EhRacG, since by mutating them with alanine, the guanine nucleotide exchange activity in vitro on EhRacG, and not on EhRho1, was affected. Moreover, we were able to obtain three candidate residues which could dictate selectivity on the EhRho1 GTPase. | |||||
BibTeX:
@Poster{A054,
author = {Hernandez Cuevas Nora A},
title = {Generation of EhGEF1 protein mutants from Entamoeba histolytica},
booktitle = {Tuesday},
year = {054}
}
|
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| Gonzalez-Andrade Martin | BIOSENSOR DESIGN ON THE PERIPLASMIC BINDING PROTEINS FOR BASIC AMINOACIDS | 055 | Monday | Poster | |
| Abstract: The biosensors are analytical tools to quantify the presence of unique molecular species from a complex mixture, as a consequence of the specific molecular-recognition by a biological macromolecule that is coupled to a transducer mechanism by physical signals. Is fundamental the development of biosensors as analytic tool for both the study of protein interactions and to the necessity to integrate the automatization of the bioanalytic systems in areas such as medicine, environmental analysis, and agriculture industry. The biosensors are conformed of two components, a biological macromolecule, which is responsible for the specific molecular-recognition and a transducer component which can report biophysical signals in a very sensitive procedure, for instance fluorescence signals. We have developed a biosensor to quantify basic amino acids by site directed labeling of the periplasmic binding protein that binds L-lisine, L-arginine L-ornithine and L-histidine (LAO protein) from Salmonella typhimurium. In this approach it was engineer by single cysteine mutation and covalently modified with a fluorescent sensitive sulfhydryl-reactive probe. The change in fluorescence emission of the protein LAO with a monobimane attached at position 167 generated a method to monitor the ligand binding process. This signal is very sensitive to monitor the free aminoacid, since quantum yield increased from 0.03 to 0.77 (25-fold) with respect to wild type protein. The biosensor developed maintained its ligand binding properties, and the same affinity to their ligands as in wild type, in the order of magnitude of nanomolar. | |||||
BibTeX:
@Poster{Martin055,
author = {Gonzalez-Andrade Martin},
title = { BIOSENSOR DESIGN ON THE PERIPLASMIC BINDING PROTEINS FOR BASIC AMINOACIDS},
booktitle = {Monday},
year = {055}
}
|
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| Guido Rafael V.C. | Structural Basis Underlying the Non-Cooperative Behavior of Glyceraldehyde-3-Phosphate Dehydrogenase from Trypanosoma cruzi | 057 | Monday | Poster | |
| Abstract: Glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi (Tc-GAPDH) is a homotetramer of four identically subunits which requires the cofactor NAD+ for activity. Tc-GAPDH catalyzes the reversible oxidative phosphorylation of D-glyceraldehyde-3-phosphate into D-glycerate-1,3-bisphosphate in the presence of the NAD+ and phosphate. The GAPDHÂs biochemical properties have been extensively investigated, however, the structural basis of the cooperativity in NAD+ binding remains to be explained. The kinetic assays revealed that Tc-GAPDH is non-cooperative upon cofactor binding. Thus, aiming at gaining structural insights into this behavior, four crystallographic structures in different states of NAD+ binding have been determined (apo, 1NAD, 2NAD, holo). The comparison between apo and holo structures indicates that the coenzyme domain undergone ~6° rigid-body rotation upon NAD+ binding. Similar results were observed for partially saturated structures 1NAD and 2NAD. The 1NAD structure can be described as a tetramer made up of three subunits in the apo conformation and the fourth subunit, containing the NAD+, adopting the holo conformation, whilst the 2NAD structure have two subunits in the apo conformation and the other two, where NAD+ molecule is bound, in the holo conformation. These results are in good agreement with the kinetic assays, since the NAD+ binding to one subunit does not disturb the overall conformation of the neighboring subunits. Moreover, the intersubunits contact analyses suggest that the Tc-GAPDHÂs non-cooperative behavior might be related to a single mutation in the tetramerÂs interface region. The information about these Tc-GAPDH forms containing sequential amounts of NAD+ contributes to shed some light on its biochemical mechanism. | |||||
BibTeX:
@Poster{V.C.057,
author = {Guido Rafael V.C.},
title = { Structural Basis Underlying the Non-Cooperative Behavior of Glyceraldehyde-3-Phosphate Dehydrogenase from Trypanosoma cruzi},
booktitle = {Monday},
year = {057}
}
|
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| Jardon-Valadez Eduardo | Modeling and Molecular Dynamics Simulation Refinement of the Human Gonadotropin-Releasing Hormone Receptor in a Lipid Bilayer | 058 | Tuesday | Poster | |
| Abstract: Since the publication of the crystal structure of bovine rhodopsin, a number of receptors belonging to the superfamily of the so called G protein-coupled receptors, have been modeled using its three-dimensional structure as the template. The resulting models have usually been validated by means of a limited number of specific interactions previously identified by mutagenesis and biochemical studies. To assess their stability, short length molecular dynamics (MD) simulations (usually in the nanosecond time scale) in vacuum or without explicit lipids have generally been performed. In the present study, a model of the human gonadotropin-releasing hormone receptor was developed, and a 160 ns long MD simulation with explicit lipid and water molecules at 310 K was used to optimize its structure. A detailed analysis of the protein structure and dynamics is presented and a number of specific contacts between different receptor amino acid residues, as well as water- and lipid-meyearted interactions are identified. The importance of ions, explicit solvent, and lipid molecules, as well as the MD simulation conditions employed proved to be critical for building a reliable model. Acknowledgments This study was supported by grants 45991-M (to A.U.-A), 41328-Q and J49811-Q (to Á.-P.) from the Consejo Nacional de Ciencia y Tecnología (CONACyT), México, and by grants 2005/1/I/159 from the FOFOI-Instituto Mexicano del Seguro Social, México, and IN105107 from PAPIIT-UNAM. |
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BibTeX:
@Poster{Eduardo058,
author = {Jardon-Valadez Eduardo},
title = {Modeling and Molecular Dynamics Simulation Refinement of the Human Gonadotropin-Releasing Hormone Receptor in a Lipid Bilayer},
booktitle = {Tuesday},
year = {058}
}
|
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| Helen Helen A Arcuri | SKPDB: a database of comparative protein strucuture models for shikimate pathway enzymes of microorganisms | 059 | Monday | Poster | |
| Abstract: The SKPDB (ShiKimate Pathway DataBase) is a relational database applied to the study of shikimate pathway enzymes in microrganisms. The main interest in the study of shikimate pathway enzymes is that its not present in humans, which make them selective targets for drug design, decreasing the impact of drugs in humans. In the database, there are currently 3185 proteins from enzymes of the shikimate pathway of microorganisms. It can be accessed on the web at http://laboheme.df.ibilce.unesp.br/skpdb/index.html or http://www.biocristalografia.df.ibilce.unesp.br/tools/index.php. Extensive information is recorded for each enzyme, including a deitaled description of the enzyme, about sequence, structure studies, functional studies, and references. All structure files are available for downloading. The current database still is in its version pilot, the data is updated regularly with the addition of new data. The models were constructed using MODELLER (a comparative protein modelling program for modelling protein structures). The overall stereochemical quality of the models were assessed by the program PROCHECK. The Root Mean Square Deviation (RMSD) between Cα Â Cα atoms distance was superposed using the program SUPERPOSE from MODELLER. The G-factor values were calculated using PROCHECK. The Verify-3D measures the compatibility of a protein model with its sequence, using a 3D profile. The modeled structures can be viewed visualization tools with Jmol. The SKPDB is a powerful tool for use in protein-ligand docking analysis. | |||||
BibTeX:
@Poster{Arcuri059,
author = {Helen Helen A Arcuri},
title = { SKPDB: a database of comparative protein strucuture models for shikimate pathway enzymes of microorganisms },
booktitle = {Monday},
year = {059}
}
|
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| Kurtenbach Eleonora | Functional and structural characterization of YER067W : an unknown ORF of Saccharomyces cerevisiae selected from a screening of hydrostatic pressure stress | 060 | Tuesday | Poster | |
| Abstract: High hydrostatic pressure (HHP) stress leads to transcriptional modifications in S. cerevisiae, however, many of the highly expressed genes are uncharacterized. Therefore, we screened for targets for structure determination by NMR of putative proteins which genes where up-regulated by HHP. The cDNA coding for 9 ORFS, YER067W, YFL014W, YDR070C, YLR327C, YMR107W, YDL110C, YPR096C, YNL266,YNL198C was inserted in pET28a and several conditions for recombinant protein expression in E. coli were tested. Inspection of the cell lysates by SDS-PAGE revealed that 7 proteins were efficiently expressed. The solubility and the aggregation state of these gene products were analyzed by gel filtration chromatography. 4 soluble proteins that eluted as a single peak in the expected retention time, according to the predicted molecular weight, were selected for structural analysis using NMR. Yer067wp presented the best 1D 1H-NMR spectrum with sharp lines and large chemical shift dispersion, characteristic of a well-structured protein. 2D [1H-15N] HSQC spectrum was recorded using uniformly labeled 15N-Yer067wp and presented all the expected HN peaks, which strongly indicates that this 20 kDa protein is suitable for structure determination using 3D NMR spectroscopy. Yer067w seems to be well conserved in fungi, however there is no close homologous with known fold or function. Working with the yeast strain deleted for YER067W we found out that those cells over accumulate glycogen and trehalose after a heat shock, suggesting that YER67W is important for the control of carbohydrate metabolism in S. cerevisiae upon stress conditions. Financial support: CNPq, PRONEX-FAPERJ. | |||||
BibTeX:
@Poster{Eleonora060,
author = {Kurtenbach Eleonora},
title = {Functional and structural characterization of YER067W : an unknown ORF of Saccharomyces cerevisiae selected from a screening of hydrostatic pressure stress},
booktitle = {Tuesday},
year = {060}
}
|
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| Henning M. Florencia | Some structural insights into the role of Apolipoprotein A-I on the neutralization of bacterial Lipopolysaccharide. | 061 | Monday | Poster | |
| Abstract: Lipopolysaccharide (LPS or endotoxin) is released from the outer surface membrane of Gram negative bacteria. LPS induces a variety of biological effects which could culminate in manifestations of septic shock. In vivo protection against endotoxin by plasma high density lipoprotein (HDL) was previously reported indicating that anti-endotoxin function of HDL was due to the interaction between LPS and apolipoproteína A-I (apoA-I), the major protein component of HDL. Effects of LPS-apoA-I interaction on the function of this protein were showed in the 1st LAPS Meeting. The aim of the present study was to investigate the interactions of LPS on the apoA-I structure. A decrease in the intrinsic fluorescence intensity of apoA-I after LPS incubation was observed which can be explained if any protein conformational changes takes place resulting in a quenching of Trp by neighbor residues. CD spectra showed changes in the environment in the region of helices of apoA-I after LPS incubation. Chemical stability was measured after incubating apoA-I and apoA-I: LPS at various [GdnHCl]. ApoA-I unfolding curve reached a plateau, and a two-model state fits the experimental data. However, LPS diminishes the cooperativity of the unfolding. G H2O was 3544 kcal/mol and 1424 kcal/mol in the absence and presence of LPS respectively, and m (coefficient proportional to the amount of hydrophobic regions of the protein exposed to solvent when the protein unfolds) decrease from 2107 to 937 in the presence of LPS. ApoA-I-LPS interaction was confirmed by photolabelling of apoAI after reaction with 125I ÂLPS ASD derivative. | |||||
BibTeX:
@Poster{Florencia061,
author = {Henning M. Florencia},
title = { Some structural insights into the role of Apolipoprotein A-I on the neutralization of bacterial Lipopolysaccharide.},
booktitle = {Monday},
year = {061}
}
|
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| Lobos-García Susana | Characterization inmunochemical of proteins by bioinformatic tools | 062 | Tuesday | Poster | |
| Abstract: The most representative variables were determined to differentiate between family proteins of binding of calcium, with the purpose of characterizing them. In this characterization the physicochemical parameters like isoelectric point were used, hydrophobicity of each protein, calculated according to 5 scales reported in literature, superficial hydrophobicity according to Hoop and Woods and the content of structures alpha and beta of studied proteins. These parameters were calculated for 65 proteins whose sequences were obtained from the PDB. In order to observe the group of the different statistical groups the method of Fisher of linear discriminant analysis was applied. Later, the function of empirical distribution was used to study the difference between the groups and thus to determine the variables with greater discriminant capacity between the studied parameters. The statistical treatment was made in the InfoStat® program. The results indicate that the variables superficial hydrophobicity (Hsuperf) and hydrophobicity of the protein calculated with according to Kyte and Dolittle (HD1), are the parameters that better allow the separation in 3 groups. In general, the data show great similarity with respect to the classification with the discriminants variables. Additionally, several antigenic methods were used to predict epitiopes with some proteins nonrelated, making a comparison between the zones predicted with the purpose of determining the most suitable methodology. | |||||
BibTeX:
@Poster{Susana062,
author = {Lobos-García Susana},
title = {Characterization inmunochemical of proteins by bioinformatic tools},
booktitle = {Tuesday},
year = {062}
}
|
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| Herrera Emma del C | Phylogenetic and conformational analysis of the HN for the Avulavirus and Rubulavirus | 063 | Monday | Poster | |
| Abstract: The hemagglutinin-neuraminidase (HN) is a common protein found in the envelope of Rubulavirus and Avulavirus. This protein has three activities: it recognizes the viral receptor on the cell surface, prevents self-aggregation of the viral progeny and promotes fusion activity between the virus and the cell. Newcastle Disease Virus (NDV) HN was crystallized in 2000 as was the Simian Virus 5 (SV-5) HN in 2005. Both structures show a barrel conformation. Taking in account the importance of the HN proteins for the viral replication cycle they may be a good target for therapeutic agents. The aim of this work was to carry out phylogenetic and structural analyses of the HN proteins for the Avulavirus and Rubulavirus. Data set were retrieved from Genpept and the alignments were carried out using Clustal W and HMMER programs. Phylogenetic analyses were realized by using the Phylip package; and for similarity analyses, the Water program from the EMBOSS package. Finally, homology models were obtained with Modeller 8v2 and evaluated with Procheck. The validation of these models was carried out with ProSa and DALI. A set of 123 sequences was chosen from Genpept, they showed a 40-60% of similarity between them. The phylogenetic tree revealed two distinct viral groups, one corresponding to Rubulavirus and other to Avulavirus genera. A high degree of conservation for the main biological domains of the HN was found by 3D homology modeling analyses, except in last  folded strips which probably is related to their affinity for different cellular receptors. | |||||
BibTeX:
@Poster{C063,
author = {Herrera Emma del C},
title = { Phylogenetic and conformational analysis of the HN for the Avulavirus and Rubulavirus},
booktitle = {Monday},
year = {063}
}
|
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| Lopez F. E. | Expression in a cell-free system of the extracellular domain of the human GABArho1 receptor | 064 | Tuesday | Poster | |
| Abstract: Gamma-amino butyric acid (GABA) is a neurotransmitter in the mammalian nervous system, whose actions are meyearted by three families of membrane receptors: GABAA, GABAB and GABAC. GABAC forms a chloride-channel constituted by pentameric associations made of one or a combination of each of three GABArho subunits (rho1-rho3); however experimental evidence has limited a full understanding of the structure of the receptor. The objective of this work was to express the domain that forms the ligand-binding site of GABArho1 in a cell-free system. A fragment coding for the 259 amino acids that forms the extracellular domain of GABArho1 was generated by PCR, cloned in-frame into the plasmid pIVEX2.3d, which allowed the fusion of a 6-HIS tag towards the C-end. Expression of the protein was performed in an rapid translation system (RTS) (Roche) at 30oC for 24h. Reactions were analyzed by PAGE and Western blot, using both an anti GABArho1 and an anti 6-HIS. Finally, affinity chromatography was used to purify the protein from the whole extract. We observed that the receptor was efficiently expressed in the conditions tested. The expressed protein was fully recognized by two independent antibodies, which assure us of the identity of the product. The affinity chromatography yielded the expected protein (40kDa), although it migrated somehow differently in PAGE. Nevertheless, it was also recognized by both specific antibodies. Further studies will try to produce high-quality protein suitable for stuctural analysis. Acknowledgments: Dra. I. Martínez-Dávila. PAPIIT 204806 and CONACYT 55025. |
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BibTeX:
@Poster{E.064,
author = {Lopez F. E.},
title = {Expression in a cell-free system of the extracellular domain of the human GABArho1 receptor},
booktitle = {Tuesday},
year = {064}
}
|
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| Lanio M.E. | Interaction of different surfactants with Sticholyins I and II (StI and StII), two toxins purified from Stichodactyla helianthus, modify their conformation and functional activity | 065 | Monday | Poster | |
| Abstract: M.E. Lanio1, C. Alvarez1, F. Pazos1, D. Martinez1, U. Ros1, M. Tejuca1, F.H. Dyszy2, S. Schreier2, E. Lissi3 1Center for Protein Studies, Faculty of Biology, University of Havana, Havana, Cuba; 2Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, Brazil; 3Faculty of Chemistry and Biology, University of Santiago de Chile, Santiago, Chile StI and StII are two toxins from the sea anemone Stichodactyla helianthus characterized by forming oligomeric pores in membranes. We have studied the conformational and functional properties of these toxins upon interaction with surfactants of different polar head group or chain length. At low SDS concentrations both toxins form aggregates, with an almost total loss of their hemolytic activity (HA). At higher SDS concentrations the protein adducts disaggregates with a decrease in fluorescence intensity (FI), loss of the native tertiary conformation and increase in alfa-helix content. However, the toxins partially recover their HA. StI and StII pre-incubation with cationic surfactants of different chain length at concentrations slightly above their CMC increases their HA and FI. The cationic surfactant effects upon HA were not found to be directly related to the changes in FI and UV-CD spectra. The increase in Sts HA observed with the cationic surfactants would indicate that association with these micelles induces changes in the toxins that favor the acquisition of a more lytically competent structure. The surfactant concentration range and the driving forces involved in surfactant-protein interaction depend both on the surfactant characteristics and the proteins structural properties. The high pI of StI and StII implies that there can be a favorable electrostatic interaction with anionic surfactants, explaining the non-cooperative effects observed for SDS at low concentrations. When cationic surfactants are considered, the association is highly cooperative and evinced only at concentrations near the surfactant CMC, a result compatible with a predominantly hydrophobic interaction. |
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BibTeX:
@Poster{M.E.065,
author = {Lanio M.E.},
title = { Interaction of different surfactants with Sticholyins I and II (StI and StII), two toxins purified from Stichodactyla helianthus, modify their conformation and functional activity },
booktitle = {Monday},
year = {065}
}
|
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| Luna Silvia S. | Modification of an amaranth seed protein and its expression in E. coli | 066 | Tuesday | Poster | |
| Abstract: Amarantin is the most predominant storage protein in Amaranthus hypochondriacus seeds; it belongs to the 11S globulin class, and shows a high content of essential aminoacids, making this protein important from a nutritional view point. This protein has two disulfide linked subunits: acidic and basic of 32-34 kDa and 22-24 kDa respectively. In order to improve the functionality of this protein, the primary structure was modified in two sites; in the globulins 11S III variable region were inserted eight aminoacids (Val-Tyr-Val-Tyr-Val-Tyr-Val-Tyr) and in the C terminal of the acidic subunit were inserted four aminoacids (Arg-Ile-Pro-Pro). Plasmids were constructed using the amarantin cDNA cloned in the pET 32b(+) expression vector, and utilizing site directed mutagenesis to insert the modified codificant sequences. There were constructed four plasmids containing the codificant sequences to produce the modified proteins: a) amarantin acidic subunit, b) internal modification of amarantin acidic subunit, c) C-terminal modified amarantin acidic subunit and, d) the protein with both modifications (internal and C terminal). They were expressed in E. coli Origami (DE3); it was observed that the acidic subunit was expressed in a higher quantity than the modified proteins, that in the proteins modified with the RIPP peptide at C terminus in the western blot assay, appear bands of high molecular weight, that may indicate that the proline inserted interacts in some way in the proper folding generating a less stable protein. | |||||
BibTeX:
@Poster{S.066,
author = {Luna Silvia S.},
title = {Modification of an amaranth seed protein and its expression in E. coli},
booktitle = {Tuesday},
year = {066}
}
|
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| Leyva Eduardo L. H. | A novel nitrilase from Rhodococcus pyridinivorans V51B that hydrolyzes aliphatic and aromatic nitriles. Cloning and over-expression of nitA gen. | 067 | Monday | Poster | |
| Abstract: Nitrilases catalyze direct conversion of organic cyanides, commonly named nitriles, to the corresponding carboxylic acids and ammonia. These enzymes are members of the carbon-nitrogen hydrolase superfamily which catalyze the hydrolysis of a wide range of non-peptide carbon-nitrogen bonds. There are no structures for the nitrilase subfamily, but the inference from sequence and structure data for two of the superfamily members is that the enzymes have a conserved, apparent catalytic triad of Glu-Lys-Cys, and fold into an α-β-β-α sandwich conformation. Bacterial nitrilases that hydrolyses aromatic nitriles as a preferred substrate have been purified and the identification of nitrilases that convert aliphatic nitriles is relatively recent. This report describes the characterization of a new nitrilase from Rhodococcus pyridinivorans and the cloning, sequencing and over-expression of the corresponding gene in E. coli. The ability of this nitrilase to hydrolyze, both, aromatic and aliphatic substrates with rather high efficiencies were observed in biotransformation assays of, whole cells and partial purified extracts. When this strain is cultivated with isovaleronitrile or ε-caprolactam, a 34 kDa band is inducible formed. The proteolytic fragments of this band were analysed by mass spectrometry using a LC-MS/MS method and identified, by highly homologous sequence in the searched database, as a nitrilase. The nitrilase gene (nitA) was amplified from the total DNA of R. pyridinivorans strain V51B; the resulting 1.1 kb product was cloned into pGEM-T and expressed in a pET vector. This nitrilase showed an amino acid identity >90% with two nitrilases of Rhodococcus rhodochrous. | |||||
BibTeX:
@Poster{H.067,
author = {Leyva Eduardo L. H.},
title = {A novel nitrilase from Rhodococcus pyridinivorans V51B that hydrolyzes aliphatic and aromatic nitriles. Cloning and over-expression of nitA gen.},
booktitle = {Monday},
year = {067}
}
|
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| Martinez-Castilla Leon P | Bayesian networks reveal coevolving amino acid that affect quaternary structure and nucleotide specificity among the betaine aldehyde dehydrogenases and related aldehyde dehydrogenases | 068 | Tuesday | Poster | |
| Abstract: Analyses of diversification patterns among proteins commonly assume that amino acids vary independently during protein evolution, but phylogeny, functional and structural constraints, and random effects can produce coevolutionary patterns. For instance, a given function may depend on the ccoperation of multiple residues, which could be replaced by by alternative combinations without affecting the phenotype. Conversely, the presence of given amino acids at certain positions may constraint the range of acceptable mutations at other positions. Betaine aldehyde dehydrogenase (BADH; E. C. 1.2.1.8) catalyzes the irreversible NAD(P)+-dependent oxidation of betaine aldehyde to glycine betaine. Biochemical studies indicate that the BADHs are a diverse group in terms of their subcellular location, physiological function, substrate specificity, coenzyme use and oligomerization state. In order to elucidate coevolutionary interactions among protein residues affecting oligomerization state or coenzyme specificity of BADHs we used Bayesian networks (BN). BNs are probabilistic models that describe statistical dependencies between multiple variables. We searched for the dependencies network that explains a maximum of correlations among positions in a BADH sequence alignment. Our model allows us to determine the specific role of residues at the BADH surface in the formation of tetrameric units and predicts the oligomerization state of certain BADHs lacking synapomorphies characteristic of tetrameric BADHs. We can also show a correlation between residues R40 and E179 from Pseudomonas aeruginosa BADH that plays a role in nucleotide specificity/versatility. Support: IXTLI-UNAM IN501506 (LPMC, RAMC), PAIP-UNAM 6290-07 (LPMC), CONACYT 45003M, DGAPA-UNAM IN224206 (HRR), DGAPA-UNAM IN206505 (RAMC). |
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BibTeX:
@Poster{P068,
author = {Martinez-Castilla Leon P},
title = {Bayesian networks reveal coevolving amino acid that affect quaternary structure and nucleotide specificity among the betaine aldehyde dehydrogenases and related aldehyde dehydrogenases},
booktitle = {Tuesday},
year = {068}
}
|
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| Luna Silvia S. | Purification and partial characterization of the acidic subunit from 11S amaranth seed protein | 069 | Monday | Poster | |
| Abstract: The most predominant storage protein in amaranth, amarantin; contains a good balance of essential aminoacids; remarkable heat stability and emulsifying properties; along with this, it has been proved that this protein is a suitable model to generate transgenic crops or to be produced in bioreactors. Moreover, this type of proteins is susceptible to protein engineering to further improve some characteristics. Amarantin acidic subunit has the potential to be employed as a nutraceutical and also as a functional protein. In order to evaluate both possibilities this protein was produced in recombinant Escherichia coli Origami (DE3) harboring the expression plasmid pET-AC6His. The protein was purified from soluble fraction in one step by immobilized metal affinity chromatography. We used two buffers: tris-HCl and phosphates, at two concentrations of NaCl; two imidazole concentrations in the binding sample buffer, and four imidazole concentrations in the elution buffer. The best results were obtained with the phosphate buffer at 0.25mM of NaCl, 10mM imidazole in the binding buffer and 100mM imidazole in the elution buffer; up to 30 mg of amarantin acidic subunit per liter of Terrific broth culture were obtained. Sucrose density gradient ultracentrifugation analysis and the HPLC analysis of the expressed soluble amarantin acidic subunit revealed that it was assembled as monomers. The His-tagged amarantin acidic subunit had an isoelectric point of 5.95; only 0.3 pH units higher than the theoretical pI. We identified this protein by Western blot and MALDI-ToF mass spectrometry. This technique proved to be a good tool for its purification. | |||||
BibTeX:
@Poster{S.069,
author = {Luna Silvia S.},
title = { Purification and partial characterization of the acidic subunit from 11S amaranth seed protein},
booktitle = {Monday},
year = {069}
}
|
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| Mecalco-Ruiz Yazmin | Electronic Structure Characterization of peptides derived from TP53-HDM2 binding domain, with possible antitumor activity. | 070 | Tuesday | Poster | |
| Abstract: Introduction. The cancer cells are deregulated in several gene pathways. The design of new antineoplasic peptide molecules to go towards to suppress or reactivates some of these gene pathways. In this study, the peptides lead to necrosis in the cancer cells. Moreover the computational quantum chemistry is useful tools that allow the design of more actives molecules. Objective. Characterize the electronic structure and the physical-chemistry properties of four synthetic peptides derived from a previously study about TP53-HDM2 binding domain. Methodology. Five peptides were analyzed (pepA-1, pepA-2, pepR-5, pepR-10, pepR-0). The geometric parameters optimization and the electronic structure calculations were carried by Gaussian 03 suite of programs. The structures were optimized at the HF level, with a 6-31G(d) basis set. The single-point calculations were performed on the optimized structure with the B3LYP/6-311G(d) level to calculated the atomic charges. Also selected geometrical parameters of the molecules were obtained to characterize the studied antitumor molecules. Finally, according to KoopmanÂs theorem, global reactivity descriptors such as the ionization potential, hardness, and the electrophilicity index were calculated. Results. The analysis of the quantum chemistry-descriptors indicated that pepA-2 and pepR-5 have smaller values of hardness, potential of ionization, and intermeyearte values of electrophilicity index. The molecular orbitals frontier (HOMO-LUMO) in the pepA-2 are located on the tryptophan, and in the pepR-5 (HOMO) it is on the tryptophan and (LUMO) on phenylalanine. Conclusions. The results atomic charges and the molecular orbitals frontier illustrate that two peptides (pepR-5 y pepA-2) analyzed were more chemistry reactivity.risso | |||||
BibTeX:
@Poster{Yazmin070,
author = {Mecalco-Ruiz Yazmin},
title = {Electronic Structure Characterization of peptides derived from TP53-HDM2 binding domain, with possible antitumor activity.},
booktitle = {Tuesday},
year = {070}
}
|
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| Silva Manzano Daniel A | In silico design of new affinities on LAO | 071 | Monday | Poster | |
| Abstract: Development in algorithms to evaluate protein stability and ligand interactions from three-dimensional structures has generated important breakthroughs in protein design. These methods offer a new scope in design of protein-protein or protein-ligand interactions. Our model system is the eriplasmic Binding Proteinfrom Salmonella typhimurium that binds L-lysine, L-arginine and L-ornithine (LAO protein). LAO belongs to a protein family with specific affinity to several ligands while sharing a common folding. Taking advantage of this characteristic, the aim of our project is to modify LAO in order to generate new protein-ligand affinities. Given the fact that this protein undergoes a conformation change towards the Âclosed state in the presence of ligand, the first phase of our project analyzed the open/closed equilibrium by molecular dynamics (MD), both in presence and absence of native ligands: arginine, lysine and histidine. In the second step, we compared the MD results obtained for physiologic ligands with its non-physiological ligands counterpart (aromatic amino acids: phenylalanine, tyrosine and tryptophan). We were able to discriminate the successful interaction of wild type LAO with native ligands versus non-native. In the third phase, we used the Rosetta program to generate in silico mutations of the amino acids around LAOÂs binding site to improve the interaction with aromatic amino acids. In the last stage we analyzed these designs by MD to find those that resemble the behavior of the wild type protein. >From these results, we believe that our simulations offer insights to choose those relevant mutations to be tested by experimentation. | |||||
BibTeX:
@Poster{A071,
author = {Silva Manzano Daniel A},
title = { In silico design of new affinities on LAO},
booktitle = {Monday},
year = {071}
}
|
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| Mejia Armando | Recombinant fusion protein containing cytochrome P450 from Amycolatopsis mediterranei and haemoglobin from Vitreoscilla stercoraria. | 072 | Tuesday | Poster | |
| Abstract: It is well known that the culture for rifamycin B production by Amycolatopsis mediterranei needs high levels of dissolved oxygen, particularly in industrial processes. In the present work, we report the expression of haemoglobin (Vhb) from Vitreoscilla stercoraria in a rifamycin B overproducing strain of Amycolatopsis mediterranei. The vhb gene was cloned under the control of the inducible PermE promoter. Industrial fermentation conditions were simulated in shake-flask cultures. Under this low aeration, the transformed A. mediterranei strain with the vhb gene showed 13.9 % higher production of rifamycin B than the parental strain in a culture with barbital, and 29.5 % under same conditions but without barbital. On the other hand, The rifamycin biosynthetic gene cluster of A. mediterranei contains five open reading frames (ORF) which are highly homologous to CYP450 monooxygenases. These proteins may be involved in some of the oxidative modification steps that occur during the biosynthesis of the rifamycin. Four of these ORFs were cloned behind T7 promoter of the bacterial expression vector pET-17b and expressed in Escherichia coli BL21 pLysS. The recombinat proteins showed typical spectral characteristics having a Soret peak at 452 nm in the reduced carbon monoxide difference spectrum. This approach allowed identification of these proteins as cytochromes P450 monooxygenases. The transcription of these five ORFs was observed during the fermentation and the effect of barbital as a possible inducer of these genes. Currently we are fusing one of these cytochrome P450 from A. mediterranei with haemoglobin from V. stercoraria. | |||||
BibTeX:
@Poster{Armando072,
author = {Mejia Armando},
title = {Recombinant fusion protein containing cytochrome P450 from Amycolatopsis mediterranei and haemoglobin from Vitreoscilla stercoraria.},
booktitle = {Tuesday},
year = {072}
}
|
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| Mate Sabina | Lipid rafts are involved in alpha-Hemolysin of E.coli oligomerization | 073 | Monday | Poster | |
| Abstract: alpha-Hemolysin (HlyA) is an extracellular protein toxin (107 kDa) secreted by Escherichia coli that acts at the level of plasma membranes of target eukaryotic cells. Considering that certain bacterial toxins utilize lipid rafts as a site for high affinity binding and oligomerization on the surface of host cells our objective was to study the role of these microdomains in the action mechanism of HlyA. Using Fluorescent Resonance Energy Transfer (FRET) technique we demonstrated that HlyA forms an oligomer on erythrocyte membranes and FRET efficiency decreases when ghost erythrocytes were cholesterol depleted with -methylcyclodextrin. Simultaneously, we found that HlyA physically associates with lipid rafts. Ghost erythrocytes were incubated with HlyA and detergent resistant membranes (DRMs) were obtained by incubation with Triton X-100 and sucrose density gradient ultracentrifugation. Immunoblot analysis and lipid characterization revealed that a substantial proportion of cell-associated toxin was associated with DRMs. Instead, ghost obtained from sheep erythrocytes treated with -methylcyclodextrin showed a significant decrease in the HlyA association with DRMs. Finally, the hemolytic activity of the toxin diminished when erythrocytes were cholesterol depleted using egg Small Unilamellar Vesicles. These results suggest the implication of lipid rafts in the oligomerization of the toxin on sheep erythrocyte membranes. | |||||
BibTeX:
@Poster{Sabina073,
author = {Mate Sabina},
title = { Lipid rafts are involved in alpha-Hemolysin of E.coli oligomerization},
booktitle = {Monday},
year = {073}
}
|
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| Mendoza Paola | Amyloid-like fibril structures developed by apolipoprotein AI derived peptides | 074 | Tuesday | Poster | |
| Abstract: Nowadays an important number of human diseases affecting several tissues and producing a series of common symptoms find their origin in the assembly of proteins into insoluble deposits. Although, the absolute establishment of this connexion is still lacking, there is solid evidence indicating a strong correlation between the formation of amyloid fibrils and their toxicity upon cells in vitro. The missing point still resides in the basic understanding of which characteristics of the so called amyloidogenic proteins define their capacity to organize themselves into a beta-structure conformation. This capacity has been on the one hand related to an hereditary component with several dominant autosomic diseases, and the other, with a Âsporadic form of the disease. Here, independently if the precursor protein is being synthesized as a normal protein, secondary external factors mainly related to the protein environment during synthesis or during transit to its target pathway, define their potential amyloidotic rute. Since not every protein that might aggregate under these conditions forms amyloidotic deposits, the study and eventually the understanding of the mechanisms that govern; first, protein folding including possible implication for disorder-to-order transitions; and second, aggregation related phenomena becomes of paramount importance. In this work we have analyzed the structural and self-assembly properties of three peptides derived from apolipoprotein AI using circular dichroism spectroscopy (CD) and atomic force microscopy (AFM). Peptide dissolution. Peptides DRV, KLL and VLES (200 mg ml-1) were prepared by dissolving them in milliQ water. Peptide solutions were incubated in polypropylene microcentrifuge tubes to prepare mature peptide fibrils. Circular dichroism spectroscopy (CD)CD ellipticity was recorded using an Aviv 60DS instrument (Aviv Instruments, Lakewood, NJ). Spectra from 260 to 190 nm were scanned at a step of 0.5 nm at 25ºC in a 0.1 cm cuvette. Atomic force microscopy (AFM).Contact mode AFM was carried out using a NanoScope III, multimode scanning force microscope and using the NanoScope III software (Version 4.23R2; Digital Instruments, Santa Barbara, CA). Peptides in solution showed to lack any secondary structure, phenomenon reversed towards an alfahelical structure when the hydrophobicity of the solution was increased, or the use of lysophospholipids in the form of micelle was standardized. Nevertheless, if peptides are maintained in water with no additions, directly related to the incubation time, peptides acquire a beta-strand structure. We show CD and AFM evidence of these transitions. | |||||
BibTeX:
@Poster{Paola074,
author = {Mendoza Paola},
title = {Amyloid-like fibril structures developed by apolipoprotein AI derived peptides},
booktitle = {Tuesday},
year = {074}
}
|
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| Matus-Garcia M. | Discovery of novel enzymatic activities in the cobalamin biosynthesis through analysis of B12 riboswitches in S. coelicolor | 075 | Monday | Poster | |
| Abstract: Cobalamin (CBL) or coenzyme B12 is one of the most chemically and structurally complex cofactors synthesized by bacteria. Salmonella typhimurium has been a model organism for the study of its anaerobic biosynthesis since the 1950s, yet the pathway is far from being completely understood. Recently it has been found that several CBL biosynthetic genes and B12-independent isozymes are regulated by riboswitches. These regulatory elements are highly conserved and widely distributed in eubacteria, and may be used for the identification of novel enzymes involved in the CBL biosynthesis in the less studied genomes. So far, CBL production has been investigated mainly in enterobacteria, and the actinomycete Streptomyces coelicolor results an interesting option since its genome is enriched with B12 riboswitches relative to that of S. typhimurium. While only two B12 riboswitches have been predicted for S. typhimurium with the RIBEX software, nine have been predicted for S. coelicolor. After analyzing the genes potentially regulated by these elements in the latter, we decided to focus on the genes of unknown function of the Sco0991-95 operon. A bioinformatic exploration and literature revision led us to suggest that the first gene of the operon, Sco0991, is an ortholog of the pduX gene in S. typhimurium and could possibly encode a threonine kinase needed in the part III of the CBL biosynthesis. The experimental confirmation of this hypothesis would highlight the usefulness of riboswitches conservation in the annotation of genes and finding of novel enzymatic activities. | |||||
BibTeX:
@Poster{M.075,
author = {Matus-Garcia M.},
title = { Discovery of novel enzymatic activities in the cobalamin biosynthesis through analysis of B12 riboswitches in S. coelicolor},
booktitle = {Monday},
year = {075}
}
|
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| Morales Ludis del Rosario | Exploratory study of the functional discrimination by 3d-plotting of isolated sea anemone neurotoxins | 076 | Tuesday | Poster | |
| Abstract: To identify the structural and functional relationships for proteins and peptides is one of the most important goals in protein and peptide studies. The purpose of this study was to generate a model based on theoretical and computational considerations. As a model to achieve this purpose previously isolated neurotoxins from sea anemones with specic actions on voltage dependant sodium and potassium channels were used. The Number of Lareo and Acevedo algorithm (NULA) was used to assign a number to each amino acid sequence of every neurotoxin tested. The NULA number obtained was then plotted using three dimensional space coordinates. The results of this study allowed us to report for the rst time that there is a different numerical and functional relationship between the sequences of amino acids from sea anemone neurotoxins and the numerical relationship for each one, or NULA number, does have a unique numerical location in a three-dimensional space. | |||||
BibTeX:
@Poster{Rosario076,
author = {Morales Ludis del Rosario},
title = {Exploratory study of the functional discrimination by 3d-plotting of isolated sea anemone neurotoxins},
booktitle = {Tuesday},
year = {076}
}
|
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| E. Meneses Hidalgo | Myb related proteins in Entamoeba histolytica genome | 077 | Monday | Poster | |
| Abstract: In this work we present the first description of the transcription factor Myb in Entamoeba histolytica. Myb proteins are highly conserved in eukaryotic organisms and their characteristic DNA-binding domain contains three tandem repeats of approximately 52 amino acids dubbed R1, R2 and R3. These repeats adopt an helix-turn-helix fold with conserved tryptophans residues. Myb proteins bind DNA sequences in a sequence specific manner and regulate expression of genes involved in cell cycle and differentiation. To identify Myb proteins in the E. histolytica genome we use c-Myb from Homo sapiens as a bait for in silico analysis in the PATHEMA E. histolytica data base and we found 18 genes with Myb domains dubbed EhMyb. EhMyb proteins from E. histolytica have 1 or 2 imperfect repeats and their phylogenetic analysis shown their organization in 3 clusters (I, II and III). Microarray analysis have shown that some Ehmyb genes suffer changes in their expression pattern during heat shock stress, enterocityc interaction and encystation, thus we propose that Myb proteins could participate in the transcriptional regulation of genes that participate in these process. In silico analysis in the promoter regions of virulence, stress response and encystation genes were performed to identify putative Myb response elements. We have produced recombinant GST fusion proteins of two EhMyb proteins and we are carrying out studies by EMSA assays to measure their binding ability with Myb response elements found in E. histolytica promoters. | |||||
BibTeX:
@Poster{Hidalgo077,
author = {E. Meneses Hidalgo},
title = {Myb related proteins in Entamoeba histolytica genome},
booktitle = {Lunes},
year = {077}
}
|
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| Noda-Garcia Lianet | Elucidating the structure/function relationship of the bifunctional phosphoribosyl isomerase (PRIA): an evolutionary approach | 078 | Tuesday | Poster | |
| Abstract: The lack of a trpF gene (tryptophan biosynthesis) in the Streptomyces coelicolor genome led to the discovery of a HisA (histidine biosynthesis) protein homologue, called PriA (phosphoribosyl isomerase). PriA is a bifunctional (β/α)8 barrel protein that catalyses the isomerisation of ProFAR (HisA susbtrate) and PRA (TrpF substrate) using the same active site. Despite the existence of a solved structure of PriA, and the high structural identity between HisA and PriA, the structure / function relationship of this enzyme remains poorly defined. Genome comparative analyses of actinomycetes showed the absence of a trpF gene and the occurrence of a trp/his operon in most of the studied microorganisms. However, this is not the case for some species belonging to the Corynebacterium genus, where the existence of an enteric trp operon (with a trpF gene), acquired by lateral gene transfer, causes the loss of PriAÂs bifunctionality. Indeed, a sequence alignment between monofunctional and bifuctional PriA / HisA homologues has pinpointed 13 residues that are different between them, suggesting their functional involvement. Independent site-directed mutageneses mimicking these naturally-occurring replacements were done on the Streptomyces coelicolor priA gene. In vivo and in vitro characterization, including X-ray crystallographic experiments leading to structure elucidation of mutants, has been performed. Results revealed specific loop recognition for each substrate and the molecular determinants underpinning the catalytic mechanism. This work represents the first example of a complete natural evolutionary history, where both, generalist (bifunctional PriA) and specialist (monfunctional PriA) enzymes have being studied. | |||||
BibTeX:
@Poster{Lianet078,
author = {Noda-Garcia Lianet},
title = {Elucidating the structure/function relationship of the bifunctional phosphoribosyl isomerase (PRIA): an evolutionary approach},
booktitle = {Tuesday},
year = {078}
}
|
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| Millan-Pacheco Cesar | TBP complexes with TATA boxes and non TATA-box containing promoters. A static and dynamic study. | 079 | Monday | Poster | |
| Abstract: The TATA-box binding protein (TBP) is used as an anchor to form the transcription pre-initiation complex even in the absence of TATA boxes. In this work, we studied the energetic and structural features of TBP-DNA complexes when TBP binds to both consensus and non-consensus TATA-box sequences. The accepted hypothesis, derived from crystallographic structures, is that TBP does not bind to guanine-cytosine containing sequences due to steric clashes at the TBP-DNA interface. In this work, i) we used 5 TBP-DNA crystallographic structures and we modified their DNA sequences in silico to estimate the energetic changes due to sequence modifications at the TBP-DNA interface, and ii) we studied the dynamic behavior of a TBP complex with a consensus and a non-consensus TATA-box sequence using molecular dynamics and free energy estimations within the MM-PBSA formalism. The results of this work show that: i) the principal contribution of the TBP-DNA binding energy is the DNA deformation energy, ii) the TBP-DNA interface can accept amino groups by forming hydrogen bonds with TBP residues, iii) we propose a method that can be used to predict TBP binding site affinity, and iv) the dynamics of TBP shows the elimination of steric clashes by deforming both TBP and DNA, as a prelude of a dissociation step of the TBP-DNA complex. | |||||
BibTeX:
@Poster{Cesar079,
author = {Millan-Pacheco Cesar},
title = { TBP complexes with TATA boxes and non TATA-box containing promoters. A static and dynamic study.},
booktitle = {Monday},
year = {079}
}
|
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| Ochoa-Leyva Adrian | Design of novel proteins through catalytic-loop exchange in TIM barrels. | 080 | Tuesday | Poster | |
| Abstract: Combining protein design and directed evolution with scaffold-based protein libraries provides an excellent route to engineer new protein function. The utility of such libraries depends on how tolerant the scaffolds are to randomization. Proteins from natural sources have already been optimized through evolution and represent an attractive starting point for the creation of novel activities. The (β/α)8 barrel proteins, in particular, are very attractive in this sense because it is a recurring scaffold in enzymes, catalyzing a variety of reactions; they are very stable, and finally, their active sites are confined to the upper part of the barrel, mainly the  loops. In this work we show a novel strategy to generate variability in proteins with a TIM barrel fold by exchanging catalytic loops among members of this family. We used the enzyme phosphorribosil antranilate isomerase (PRAI) as a template to generate novel proteins leading to evolution of new functions. We investigated the tolerance of PRAI to loop exchange. Variability was allowed in the residues that point towards the interior of the barrel and precede the exchanged loop. We describe the exchange of 13 different (β/α) loops within the PRAI structure. The viability of the constructions was evaluated by fusing them to a folding reporter gene. The high percentage of folded chimeras with exchanged loops in the catalytic face of PRAI confirmed the robustness of this fold. The results obtained were compared to a theoretical prediction of the variant stabilities using the ΔΔG Rosetta and Rosetta Design algorithms on the expected libraries. | |||||
BibTeX:
@Poster{Adrian080,
author = {Ochoa-Leyva Adrian},
title = {Design of novel proteins through catalytic-loop exchange in TIM barrels.},
booktitle = {Tuesday},
year = {080}
}
|
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| Morett Enrique | The promiscuous enzymatic activity of the E. coli proteome as a source of new protein function | 081 | Monday | Poster | |
| Abstract: Enzymes are highly efficient and selective catalysts. However, due to the intrinsically reactive nature of the active sites, secondary, normally negligible activities are practically unavoidable and have been observed in many enzymes. Consequently, the proteome of a bacterial cell has the potential to carry out a great deal of secondary activities, which could be the basis of new enzymes in the process of natural evolution. To test for the ability of E. coli proteome to perform an arbitrarily selected reaction by a secondary enzymatic activity, we used a very sensitive selection method based in growth complementation of thiamin deficient strains. By overexpresing the cellś protein repertoire we selected yjbQ, a gene with not naturally selected secondary thiamin phosphate syntase (TPS) activity. Structurally oriented mutagenesis and directed evolution for enhanced TPS activity allowed us to identify the putative active site. Orthologs of very distantly related species also have TPS activity, suggesting that the secondary TPS activity is associated with the conserved active site. Our results support the view that the natural enzymatic repertoire has the potential to perform vast amounts of secondary reactions, which could be exploited as starting points to evolve new or more efficient catalysts. | |||||
BibTeX:
@Poster{Enrique081,
author = {Morett Enrique},
title = { The promiscuous enzymatic activity of the E. coli proteome as a source of new protein function},
booktitle = {Monday},
year = {081}
}
|
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| Peimbert Mariana | PRAI thermostabilization by consensus-based engineering | 082 | Tuesday | Poster | |
| Abstract: We constructed a stabilized N-[5´-phosphoribosyl] anthranilate isomerase from Escherichia coli (ePRAI) by recruitment of consensus mutations. We aligned 48 PRAI sequences (mostly from pathogenic bacteria), 44 positions where E. coli gene differs from the 30% consensus were identified. ePRAI is one of the smallest (beta/alpha)8-barrel proteins known, for which kinetic and equilibrium folding intermeyearte has been described. The consensus enzyme (conPRAI) showed a 17 degrees C increase in unfolding temperature compared to ePRAI. The second unfolding transition for conPRAI was increased in 3.97 kcal/mol. Since the total Delta G was increased only by 2.5 kcal/mol, we conclude that the folding intermeyearte was stabilized. | |||||
BibTeX:
@Poster{Mariana082,
author = {Peimbert Mariana},
title = {PRAI thermostabilization by consensus-based engineering},
booktitle = {Tuesday},
year = {082}
}
|
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| Munoz-Clares Rosario A | Incubation with Ligands Induces Active Site Heterogeneity in the Dimeric Betaine Aldehyde Dehydrogenase from Plants | 083 | Monday | Poster | |
| Abstract: The chloroplastic betaine aldehyde dehydrogenase (chBADH) catalyzes the irreversible NAD+-dependent oxidation of betaine aldehyde (BA) producing the osmoprotectant glycine betaine. The enzyme is a homodimer which exhibits an Iso Bi Bi Ordered Steady-State mechanism. Both BA and NAD+ produce substrate inhibition. In this work we report the partial inactivation of chBADH by incubation with BA or NADH under non-catalytic conditions. The extent and the rate of inactivation depended on ligand concentration in a hyperbolic manner, showing saturation. The results are consistent with a slow, reversible conformational change induced by the ligands upon binding to the enzyme, leading to an inactive enzyme form. Reactivation in the assay medium was observed when the reaction was followed during an extended period of time. Pre-steady state kinetics indicated that the enzymes pre-incubated with BA (PreincBA) or NADH (PreincNADH) and the non-pre-incubated (control) enzyme had an activity burst, suggesting that the rate-limiting step takes place after hydride transfer. But whereas the control enzyme showed full sites reactivity, the PreincBA and PreincNADH enzymes exhibited half of the sites reactivity. Steady-state studies showed apparent negative cooperativity in the saturation kinetics of NAD+, suggesting that the two sites are active, but one of them has a much decreased affinity for NAD+. Saturation by NADH also exhibited apparent negative cooperativity in the PreincNADH enzyme but apparent positive cooperativity in the PreincBA enzyme. Moreover, NADH appears to be a total inhibitor of the former and a partial inhibitor of the latter. Supported by DGAPA-UNAM grant IN206505 |
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BibTeX:
@Poster{A083,
author = {Munoz-Clares Rosario A},
title = {Incubation with Ligands Induces Active Site Heterogeneity in the Dimeric Betaine Aldehyde Dehydrogenase from Plants},
booktitle = {Monday},
year = {083}
}
|
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| Plumbridge Jacqueline A | In silico modelling and characterization of the putative GlcNAc6P binding site of NagC of E.coli | 084 | Tuesday | Poster | |
| Abstract: NagC, is a transcriptional regulator which co-ordinates the metabolism of aminosugars in E.coli. It is a member of the so-called ROK family and its effector molecule was previously identified as GlcNAc6P. NagC is homologous to Mlc, the repressor of the Glucose PTS, whose 3D structure is known (IZ6R). Although Mlc does not bind glucose it has structural homology to E.coli glucose kinase. The residues ressembling the glucose binding site are conserved in NagC and mutagenesis of three of these five positions, H194, E244 and E266, produced non-inducible forms of NagC. We have modelled the structure of NagC (using Modeller) based on that of the four crystallographic chains of Mlc and a ROK protein from Vibrio cholerae (IZ05). H194 and E244, E266 lie on either side of a cleft between two domains of the protein. Docking of GlcNAc6P (using Autodock3) indicates a binding site in the cleft such that binding of GlcNAc6P could be expected to change the relative positions of the two domains and hence that of the DNA binding domain. Docking GlcNAc6P to open and closed forms of Mlc suggests the participation of several other amino acids in GlcNAc6P binding such as R95. This residue is predicted to bind the phosphate motif of GlcNAc6P. The mutations R95K, H194A, E244A, G246A and E266A were generated in silico and GlcNAc6P was docked to each one of them. The results strongly correlate with the experimental data lending further support to the putative GlcNAc6P binding site of NagC. | |||||
BibTeX:
@Poster{A084,
author = {Plumbridge Jacqueline A},
title = {In silico modelling and characterization of the putative GlcNAc6P binding site of NagC of E.coli},
booktitle = {Tuesday},
year = {084}
}
|
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| Nicoluci R. P. | Investigation of human Adenylosuccinate lyase mutations by structural, kinetic and Stastistical Coupling Analysis. | 085 | Monday | Poster | |
| Abstract: Adenylosuccinate lyase (ADSL) is an enzyme that plays a critical role in both cellular replication and metabolism via its action in the purine biosynthetic pathways. ADSL is the only enzyme in this pathway to catalyze two separate reactions, enabling it to participate in the addition of nitrogen at two different positions in adenosine monophosphate. Because ADSL plays an integral part in maintaining proper cellular metabolism, mutations in the human enzyme have severe clinical consequences, including mental retardation with autistic features. Several point mutations in ADSL have been identified that are associated with deficiency of enzymatic activity and either psychomotor retardation or autistic features. Seven of them (Y114H, R190Q, R194C, D268N, L311V, P318L, D430N) were performed and had their kinetic parameters established. Considering its relevance, the purpose of this study is to associate these point mutations with the kinetic properties trough the structural modification. After validation of 3D structure of human ADSL made by molecular modeling techniques, these seven point mutations were structurally evaluated and compared to their kinetic values. Also a Statistical Coupling Analysis (SCA) was performed and could identify that mutations with severe symptoms belong to an evolutionary and inter-connected pattern of amino acids. This analysis can bring new insights into the understanding of the structure  function correlation on ADSL activity. | |||||
BibTeX:
@Poster{P.085,
author = {Nicoluci R. P.},
title = { Investigation of human Adenylosuccinate lyase mutations by structural, kinetic and Stastistical Coupling Analysis.},
booktitle = {Monday},
year = {085}
}
|
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| Portillo-Tellez Maria del Carmen | Denaturation and Renaturation of Aldolase in the Presence of Compatible Solutes | 086 | Tuesday | Poster | |
| Abstract: Anhydrobionts are organisms that use compatible solutes to survive dessication. Compatible solutes show a great potential to be used as protein stabilizers. In this study the compatible solute trehalose was used to increase the stability of rabbit muscle aldolase during folding-unfolding caused by different concentrations of guanidine HCl. Aldolase is an homotetrameric enzyme that participates in glycolisis and gluconeogenesis. After a cycle of denaturation/renaturation, aldolase recovered its activity and secondary and tertiary structure to different degrees depending on the concentration of trehalose. This was assessed by activity, fluorescence and CD assays. Aldolase deactivation was complete in the presence of guanidine HCl. Trehalose did not protect the enzyme during denaturation, however, when trehalosa 0.6 M was added during denaturation, the enzyme could be completely reactivated upon 100 fold dilution. By contrast, if high trehalose concentration was maintained during renaturation/reactivation, this process became very slow. These data are were compatible with a trehalose meyearted inhibition of the intramolecular vibration of the protein during refolding. | |||||
BibTeX:
@Poster{Carmen086,
author = {Portillo-Tellez Maria del Carmen},
title = {Denaturation and Renaturation of Aldolase in the Presence of Compatible Solutes},
booktitle = {Tuesday},
year = {086}
}
|
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| Ordoñez Leandro G | Modeling, construction and bioactivity of chimeric proteins of the human interferon gamma and the human interleukin-2 | 087 | Monday | Poster | |
| Abstract: The recombinant human interferon-gamma (hIFN-g) and the recombinant human interlukin-2 (hIL-2) are used simultaneously in the therapy of the renal carcinoma. These cytokines are expensive, thus the design of chimeras let reduce the production cost. In this work, the hIFN-g and hIL-2 synthetic genes were connected by a synthetic linker encoding poly-glicine and the chimeric gene was expressed in Escherichia coli. Two different fusions proteins were constructed, consisting in hIFN-g:hIL-2 and hIL-2:hIFN-g variants. An additional construction named as hIFN-g:hIL-2m was obtained by introducing the mutations T159S and K216E in the hIFN-g:hIL-2 sequence. The structure of the chimeras was forecasted by computational modeling and bioactivity of the hIFN-g and hIL-2 in the same molecule was evaluated. The effect of hIFN-g was measured as expression of major histocompatibility complex class I (MHC-I) molecules on the pre-monocytes U937 cell line, whereas the proliferative activity of the hIL-2 was assessed on lymphocytes T CTLL-2 cell line. The bioactivities of the chimeras were compared using recombinant standards of hIFN-g and hIL-2 alone and in equivalent combination as controls. Both hIL-2:hIFN-g and hIFN-g:hIL-2m chimeras were bifunctional, they exhibited increasing expression of the MHC-I molecules in U937 cells and proliferative activity in CTLL-2 cells, but not the hIFN-g:hIL-2. The results suggest that the hIL-2:hIFN-g and the hIFN-g:hIL-2m fusion proteins could be future promise as therapeutic agents in the treatment of renal carcinoma where stimulation of immune system and proliferation of T cells are required. Acknowledgments: This work was partially supported by CONACyT-SALUD grant 05-13993 | |||||
BibTeX:
@Poster{G087,
author = {Ordoñez Leandro G},
title = { Modeling, construction and bioactivity of chimeric proteins of the human interferon gamma and the human interleukin-2},
booktitle = {Monday},
year = {087}
}
|
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| Primo Maria E. | Crystal structure of the mature extracellular domain of IA-2 unveils similarity to mucins | 088 | Tuesday | Poster | |
| Abstract: The 106-kDa insulinomaÂassociated protein (IAÂ2), a member of the receptor protein tyrosine phosphatase superfamily, is located in the membrane of secretory granules (SGs) of neural, pituitary and pancreatic islet cells. It became known because it is related to insulin secretion and autoimmunity disorders in yearbetes. IA-2 is post-translationally processed by proteolytic cleavage, yielding a mature extracellular domain (meIA-2, residues 449-576), attached to the naturally inactive intracellular domain (IA-2ic) through a single transmembrane region (residues 576?600). In a previous work we reported that meIA-2 is an autonomous folding unit and now we determined its 3D structure by X-ray diffraction with 1.3 Å resolution. Unexpectedly, the solved structure only includes residues 469-557, which was confirmed by mass analysis. Further investigation demonstrated that auto proteolysis of the chain extremes takes place during crystallization. The fragment of meIA-2 has a b1-a1-b2-b3-a2-b4 fold, in which the helices rest on top of a beta sheet. This fold correspond to the SEA domain typical of glycosylated extracellular domains, like those found in mucins. MeIA-2 was found to dimerize in solution, and in the crystal, meIA-2 exhibits two different dimerization modes that might be relevant in vivo and permit the its interaction with a plethora of extracellular moieties and membranes. This work was supported by grants from Conicet, UNQ, UBA, and ANPCyT. |
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BibTeX:
@Poster{E.088,
author = {Primo Maria E.},
title = {Crystal structure of the mature extracellular domain of IA-2 unveils similarity to mucins},
booktitle = {Tuesday},
year = {088}
}
|
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| Padilla-Benavides Teresita Del N.J. | Interaction between b-subunits of the na+, k+ atpase in-vivo: fret assay. | 089 | Monday | Poster | |
| Abstract: Sodium pumps belonging to two neighboring cells interact across the intercellular espace through their B-subunits (Shoshani et al., 2005). We now resort to protein-protein interaction assays in-vitro that allow us to demonstrate that a B-subunit is able to associate with another B-subunit in a specific manner. Nevertheless, with these biochemical methods we can not state clearly if the B-B interaction is established among two B-subunits of the same cell or between the membranes of two neighboring cells. For clarifying this issue, we are planning to study protein interactions by FRET microscopy in living cells. Therefore we will tag the B-subunit of the Na+, K+ ATPase with the yellow fluorescent protein (YFP) and other with cyan fluorescent protein (CFP) and express them in mammalian cells. These cells will be co-cultured and heterotypic contact membranes analyzed by FRET. If the B-subunits of two neighboring cells are associated, we will be able to calculate the efficiency of energy transfer and relate it to the distance between B-subunits. So far, we have cloned the rat B1-subunit in both vectors (YFP and CFP), we transfected N-terminal fused YFP-B1-subunit in CHO cells and find that the expression is enriched in the plasma membrane between contacting cells. However, in order to measure the FRET efficiency between the pair CFP-YFP we need to express the CFP-B1 subunit in the membrane of CHO cells. | |||||
BibTeX:
@Poster{N.J.089,
author = {Padilla-Benavides Teresita Del N.J.},
title = {Interaction between b-subunits of the na+, k+ atpase in-vivo: fret assay.},
booktitle = {Monday},
year = {089}
}
|
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| Ramirez-Diaz Martha | Expression and purification of the chromate resistance Chrb protein from pseudomonas aeruginosa | 090 | Tuesday | Poster | |
| Abstract: Introduction. The chr operon from the pMOL28 plasmid which confers resistance to chromate in Cupriavidus metallidurans, includes the chrA, chrB, chrC, chrE and chrF genes (1). The chrB gene encodes the ChrB protein that has been suggested to function as a transcriptional regulator of chromate resistance, since its absence leads to hypercapture and hypersensitivity to this ion (1, 2). A recent sequence analysis of the pUM505 plasmid from Pseudomonas aeruginosa revealed a chrB putative sequence upstream of chrA, with 45% amino acid identity with its pMOL28 homolog; the precise function of chrB is still unknown (3). The objectives of this work are the expression and purification of ChrB from pUM505 and to evaluate its possible participation in the regulation of the resistance to chromate. Material and methods. The open reading frame of chrB lacking the initiation codon was amplified by PCR. The PCR fragment was first ligated into the pQE31 expression vector that adds a 6-His tag and the recombinant plasmid was transferred into Escherichia coli BL21. Expression of the chrB gene was tested at different conditions: induction with 0.2, 0.5, 1 and 2 mM of IPTG, growth for 4 or 12 h, and incubation at 30 or 37oC. Expression analysis was made by SDS-PAGE of cell extracts. The ChrB-His protein was detected by Western blot assays using a His-Probe reagent. ChrB-His purification was performed by using a Ni-NTA affinity chromatography method. Results. The overexpression of the ChrB recombinant protein was achieved after incubation of cultures for 4 h at 28°C with the addition of 1.0 mM IPTG. ChrB showed a ~32-kDa molecular mass in SDS-PAGE. Western blot assays confirmed that the major band corresponded to the ChrB protein. Finally, ChrB was purified following elution from a Ni-NTA column. Discussion. The predicted size of the translation product of the chrB gene was consistent with the ~32-kDa size of the polypeptide overexpressed after IPTG induction. The signal visible in the Western blot autoradiograms also corresponded to the ~32-kDa protein, confirming that this polypeptide is the ChrB protein. Purified ChrB is currently being used in assays of DNA-protein interactions to evaluate the possible function of this protein in the chromate resistance phenotype. Bibliography 1. Juhnke S. et al. (2002) Arch. Microbiol. 179:15-25. 2. Nies A. et al. (1990) J. Biol. Chem. 265:5648-5653. 3. Díaz A. (2006) Thesis. Faculty of QFB, UMSNH. This work was supported by grant CB0702157_0 from the COECYT (México). |
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BibTeX:
@Poster{Martha090,
author = {Ramirez-Diaz Martha},
title = {Expression and purification of the chromate resistance Chrb protein from pseudomonas aeruginosa},
booktitle = {Tuesday},
year = {090}
}
|
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| Pastor-Palacios Guillermo | DNA polymerase I of Entamoeba histolytica | 091 | Monday | Poster | |
| Abstract: E. histolytica was classified as amitochondriate parasitic protozoa, but recent studies indicate the presence of mitochondrial genes and the presence organelles that contain DNA. However, the presence or absence of genetic material in these organelles is not well defined, but the idea that E. histolytica evolved from a mitochondriate ancestor and secondary loss of the mitochondria is accepted. Family A polymerases are involved in the DNA replication of extranuclear DNA in protozoan parasites, as Plasmodium falciparum and Trypanosoma cruzi, thus we formulate the hypothesis that a family A DNA polymerase maybe involved in the replication of extranuclear DNA in E. histolytica. In this work we present, the cloning of the DNA polymerase gene from E. histolytica in a expression vector. With regard to the biochemical characterization of the DNA polymerase I from E. histolytica, we were able to purify the recombinant protein using nickel affinity chromatography, we found that the protein was active in a primer extension assay and displayed affinity to double stranded DNA. The DNA polymerase gene is expressed at the level of messenger RNA, as tested by RT-PCT assay, and the protein is translated and it is found both in the cytoplasm as was shown by a Western-Blot assay using polyclonal antibodies against a peptide of the DNA polymerase of E. histolytica. Because of the cellular localization of the DNA polymerase of E. histolytica, we think that is possible that this polymerase has a role in the replication of extra nuclear DNA in E. histolytica. | |||||
BibTeX:
@Poster{Guillermo091,
author = {Pastor-Palacios Guillermo},
title = { DNA polymerase I of Entamoeba histolytica},
booktitle = {Monday},
year = {091}
}
|
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| Reyes-Lopez C.A. | Chemical unfolding of Saccharomyces cerevisiae enolase | 092 | Tuesday | Poster | |
| Abstract: The unfolding reaction of enolase from Saccharomyces cerevisiae (yeast enolase) induced by guanidine hydrochloride (GdnHCl) was investigated by fluorescence spectroscopy. This homodimeric enzyme (46.7 kDa per subunit) catalyses the reversible dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate. Enolase is an interesting model for folding/unfolding studies because of its structure and its involvement in the glycolytic pathway. Each monomer of yeast enolase consist of two domains; the largest one displays an /-barrel scaffold and the smallest domain provides a loop which closes over the active site upon substrate binding. Fluorescence-emission spectra of protein solutions (0.2 M, on monomer term), incubated at several denaturant concentrations from 0 to 6M, were obtained and their spectral centers of mass (SCM) were calculated. Transition curves were built by plotting the SCM values vs. GdnHCl concentration. As judged from the transition curves, the unfolding reaction of yeast enolase involves at least one intermeyearte, under the experimental conditions used. The denaturation profiles were analyzed by an equation that describes a three-state model for unfolding. From this analysis the apparent thermodynamic parameters associated to the unfolding reaction were calculated. Dilution-induced refolding assays showed that yeast enolase denaturation is not fully reversible. Additionally, we also present an analysis of the effect of the phosphate-ion on the GdnHCl-induced unfolding of yeast enolase. Results showed that the phosphate group stabilizes the native and intermeyearte forms of the enzyme without changing the unfolding mechanism. | |||||
BibTeX:
@Poster{C.A.092,
author = {Reyes-Lopez C.A.},
title = {Chemical unfolding of Saccharomyces cerevisiae enolase},
booktitle = {Tuesday},
year = {092}
}
|
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| Pedraza-Escalona M. Martha | Two B-cell epitopes of the allergen Hev b 6.02. Molecular analysis revealed by the use of murine monoclonal antibodies and human single chain antibody fragments. | 093 | Monday | Poster | |
| Abstract: Introduction IgE-meyearted reactions (type I hypersensitivity) to Natural rubber latex (NRL) have become a widely recognized medical problem among individuals who are in contact with allergenic proteins found in products manufactured with this material. Hev b 6.02 (hevein), the major NRL allergen, is a 4.7-kDa lectin that also acts as a pathogenesis defense-related protein. Several analysis of a variety of plant-derived food allergens have resulted in the identification of this polypeptide chain as a chitin-binding domain in class I endochitinases, which has explained the cross-reactivity between latex and some fruits allergy. The linear epitopes of Hev b 6.02 have been analized, although the information about conformational epitopes is still lacking for an accurate treatment as well as for the study of molecular interaction allergen-antibody. Objective We sought to study at a molecular and structural level two B cell epitopes of Hev b 6.02, analized by specific mAbs and scFv to this allergen. Methods Hev b 6.02 specific-murine monoclonal antibodies were obtained by immunization and human single chain fragments variable was isolated from a non-immune phage display library. Isoforms and modified chemically hevein were used to localize the binding sites. Furthermore intrinsic fluorescence measurements of tryptophan residues and acrylamide quenching. Simulated docking experiments to fit hevein into the paratope region of both types of recombinant antibodies. Results The human antibody and several anti-Hev b 6.02 monoclonal antibodies were shown to inhibit IgEs (from sera of natural rubber latex patients) binding to Hev b 6.02 in different percentage. Two murine monoclonal anti-Hev b 6.02 antibodies (mAb 6E7 and 6D6) defined the tryptophans area of Hev b 6.02 like one IgE epitope. Whereas a Hev b 6.02 specific-scFv defined the C-terminal region of this allergen like other IgE epitope. | |||||
BibTeX:
@Poster{Martha093,
author = {Pedraza-Escalona M. Martha},
title = { Two B-cell epitopes of the allergen Hev b 6.02. Molecular analysis revealed by the use of murine monoclonal antibodies and human single chain antibody fragments.},
booktitle = {Monday},
year = {093}
}
|
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| G. Rodríguez | Expression of a recombinant antigen (Ov16) to Onchocerca volvulus. | 094 | Martes | Poster | |
| Abstract: The elimination onchocerciasis program in Mexico is based entirely on the use of ivermectin a drug that kills the skin microfilariae but not the adult worms. Thus, is important to carry out prospective entomological studies to evaluate the effect of ivermectin on transmission levels, and performing the monitoring for new infections in children, parasitologically or serologically, as an indicator of new infections. Although several antibody detection tests for onchocerciasis have been developed, the programs require accurate, sensitive, non-invasive, and 100% specific tests for onchocerciasis. A serologic test based on the use of a recombinant antigen (Ov16) in an ELISA protocol (using a conjugate to test for IgG4) is currently used by the onchocerciasis programs in Mexico and Guatemala. Given that the test requires Ov16 the objective of this study was to produce the recombinant protein using the binding protein maltose system. The production of the recombinant proteins was optimized, the resultant proteins were dialysed to remove any traces of contaminating maltose, and the final concentration was determined using the Bradford technique. The optimum time to obtain the highest yield was three hours after induction with IPTG with an average yield (n= 2) of 0.7 mg/ml per liter of culture. A total of 1.162 mg/ml of recombinant antigen was obtained, and it is estimated that 3500 serologic samples may be tested with this amount of protein. Additional production experiments are being conducted to provide this recombinant antigen as much as needed by the onchocerciasis programs. | |||||
BibTeX:
@Poster{,
author = {G. Rodríguez},
title = {Expression of a recombinant antigen (Ov16) to Onchocerca volvulus.},
booktitle = {Martes},
year = {094}
}
|
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| Perez Medina Martinez Victor | Mapping of protein interaction domains between two major ribonucleases of Escherichia coli. | 095 | Monday | Poster | |
| Abstract: Introduction. The RNA Degradosome of Escherichia coli is a complex formed by RNaseE, RhlB helicase, Enolase and PNPase enzymes. It is known that its resident enzymes could be exchanged under some grow conditions. RNasePH has similar tertiary structural features similar to PNPase, and it has been demonstrated that complexes between RNaseE and RNasePH could exists in vivo using a Âpull down assayÂ. Methods. Double hybrid assay was used to map the domains of interaction of RNasePH with RNaseE in vivo detected as Streptomycin resistance and survival on minimal medium. RNasePH was divided in five sections of eighty overlapping aminoacids; and each section was cloned in the bait and RNaseE in the target vector. A reciprocal test was also made. Results. After transforming Bacteriomatch-II with the plasmid libraries; clones containing rph fragments (001-080aa) were isolated in both tests from bacteria growing on M9 minimum medium with antibiotics and 4.5 mM 3-amino-1,2,4-triazol as inhibitor of His3. Streptomycin was used as a second reporter for selection. DNA plasmid of six candidate clones were purified and sequenced for analysis. Conclusions. An RNasePH domain composed of four beta-sheets found in the first eighty aminoacids was identified as the interaction domain with RNaseE. This led us to propose that in the maturation of stable RNAs, an RNasePH-RNaseE complex might participate. A complex where RNasePH substitutes dynamically PNPase in the degradosome could be formed under certain growth conditions. Work financed for a UCMexus-Conacyt grant to JGM. | |||||
BibTeX:
@Poster{Victor095,
author = {Perez Medina Martinez Victor},
title = { Mapping of protein interaction domains between two major ribonucleases of Escherichia coli.},
booktitle = {Monday},
year = {095}
}
|
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| Rodriguez-Vargas Victor H. | Co-expression with chaperones favor the expression in a soluble fraction of the cisteine-proteinase TVLEGU-1 of trichomonas vaginalis. | 096 | Tuesday | Poster | |
| Abstract: ABSTRACT: TVLEGU-1 is an asparaginyl endopeptidase, the first cysteine proteinase (CP) of clan CD identified in the 30-kDa region involved in the Trichomonas vaginalis cytoadherence. As other cysteine proteinases involved in T. vaginalis virulence, gene expression, and proteolytic activity of TVLEGU-1 is regulated by iron concentrations, suggesting that it may participate in parasite virulence. The aim of this work was to express a soluble recombinant TVLEGU-1 in order to perform functional and structural studies. The 1,164-bp ORF of the tvlegu-1 gene was cloned into the pCold I expression vector and expressed in Escherichia coli. To obtain the recombinant protease in soluble form, we expressed the TVLEGU-1 through the co-expression of pCold I vector and the chaperone team plasmids pG-Tf2, pTf16, pGro7 or pKJE7. By SDS-PAGE and Western blot assays with anti-TVLEGU-1 antibodies a 43-kDa band of the recombinant TVLEGU-1 was found only in the insoluble fraction when E. coli was transformed by the pCold I construct alone. The co-expression of TVLEGU-1with GroEL-GroES-Tig (pG-Tf2) or with Tig (pTf16) favors the expression as a soluble recombinant protein. | |||||
BibTeX:
@Poster{H.096,
author = {Rodriguez-Vargas Victor H.},
title = {Co-expression with chaperones favor the expression in a soluble fraction of the cisteine-proteinase TVLEGU-1 of trichomonas vaginalis.},
booktitle = {Tuesday},
year = {096}
}
|
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| Piña Edgar O. | In silico exploration of the sequence space within Cysteine Stabilized alpha/beta motif | 097 | Monday | Poster | |
| Abstract: Invertebrate defensins are the most widely spread class of antibiotic peptides, with i dentified members in plants and several metazoans, including nematodes, mussels and arthropods. Structurally, they adopt the so-called Cysteine Stabilized alpha/beta (CS-alpha/beta) motif. This structural motif is also adopted by other functionally diverse groups of disulphide rich peptides, amongst which scorpion toxins are the largest and more diversified group, accounting for hundreds of identified members. Compiling evidence about the three-dimensional folding, gene topology and the hyperdiversified family of scorpion venom peptides Âlikely derived from paralogous expansionsÂ, it has been hypothesized that scorpion toxins and invertebrate defensins are homologous groups, with the antimicrobial peptides being the ancestral group. Actually, scorpionsgenomes encode for both kinds of groups; however, the hyperdiversified paralogous nature of scorpion toxins does not allow resolving the putative evolutionary relationships between both groups. In this work we try to resolve whether or not the sequence space of invertebrate defensins can support the derived function of scorpion toxins (K+ channel blockade). Based on a common strategy of protein engineering, we identified a suitable defensin sequence and, by in silico modeling, grafted the molecular determinants of the K+ channel blockade function displayed by the derived group (the scorpion toxins). Several variants were then assayed by soft-docking procedures, in order to identify if they are likely to interact with K+ channels. These analyses were then used to design chimeric peptides, which are being tested for their antimicrobial (ancestral) and k+ channel blocking (derived) activities. | |||||
BibTeX:
@Poster{O.097,
author = {Piña Edgar O.},
title = {In silico exploration of the sequence space within Cysteine Stabilized alpha/beta motif},
booktitle = {Monday},
year = {097}
}
|
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| Rojas Adriana L | Functional architecture of the retromer cargo-recognition complex | 098 | Tuesday | Poster | |
| Abstract: The retromer complex is required for the sorting of acid hydrolases to lysosomes, transcytosis of the polymeric immunoglobulin receptor, Wnt gradient formation, iron transporter recycling and processing of the amyloid precursor protein. Human retromer consists of two smaller complexes: the cargo recognition VPS26ÂVPS29ÂVPS35 heterotrimer and a membrane- targeting heterodimer or homodimer of SNX1 and/or SNX2. Here we report the crystal structure of a VPS29Â VPS35 subcomplex showing how the metallophosphoesterase-fold subunit VPS29 acts as a scaffold for the carboxyterminal half of VPS35. VPS35 forms a horseshoe-shaped, righthanded, a-helical solenoid, the concave face of which completely covers the metal-binding site of VPS29, whereas the convex face exposes a series of hydrophobic interhelical grooves. Electron microscopy shows that the intact VPS26ÂVPS29ÂVPS35 complex is a stick-shaped, flexible structure, approximately 21nm long. A hybrid structural model derived from crystal structures, electron microscopy, interaction studies and bioinformatics shows that the a-solenoid fold extends the full length of VPS35, and that VPS26 is bound at the opposite end from VPS29. This extended structure presents multiple binding sites for the SNX complex and receptor cargo, and appears capable of flexing to conform to curved vesicular membranes. Accepted for publication in Nature |
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BibTeX:
@Poster{L098,
author = {Rojas Adriana L},
title = {Functional architecture of the retromer cargo-recognition complex},
booktitle = {Tuesday},
year = {098}
}
|
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| Quintas-Granados Laura I. | EhCP112 a cysteine proteinase involved in Entamoeba histolytica virulence: precursor activation and inhibition studies. | 099 | Monday | Poster | |
| Abstract: Cysteine proteases are known to be important pathogenicity factors for Entamoeba histolytica. EhCP112 is a cysteine proteinase that assembles with the adhesin EhADH112 to form the EhCPADH complex involved in E. histolytica virulence. In a previous work, the recombinant EhCP112 precursor was expressed in E. coli as inclusion bodies and purified under denaturing conditions. To test the effect of different peptidase inhibitors, first the unfolded precursor (52 kDa) was refolded by a gel filtration chromatography, and then the 52 kDa inactive polypeptide was incubated under reduction conditions to obtain the active mature EhCP112. The active mature enzyme was incubated with several peptidase inhbitors and with the recombinant EhCP112 Âpre-pro fragment and after 10 min incubation the residual enzyme activity was determined by a spectrophotometric assay and zymograms using azocasein and gelatin as substrates respectively. Specific inhbitors for serine, aspartic or metallo proteinases did not inhibited EhCP112 activity. In contrast, enzymatic activity of rEhCP112 was greatly reduced or completely inhibited with all cysteine proteinase inhibitors tested such as E64, leupeptin, chymostatin, antipain and TLCK and with five molar excess of its recombinant Âpre-pro fragment. The cysteine proteinase inhibitors also inhibited the activation of EhCP112 precursor. EhCP112 unfolded inactive precursor can be refolded and auto processed to mature active enzyme under reduction conditions. EhCP112 proteolytic activity is inhibited by cysteine proteinase inhibitors and by its own Âpre-pro fragment. This work was supported by CONACYT-MEXICO grant number 40387-Z. | |||||
BibTeX:
@Poster{I.099,
author = {Quintas-Granados Laura I.},
title = { EhCP112 a cysteine proteinase involved in Entamoeba histolytica virulence: precursor activation and inhibition studies.},
booktitle = {Monday},
year = {099}
}
|
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| Rosas-Trigueros Jorge L | Protein Structure Prediction using Dynamic Semi-Exhaustive Conformational Search | 100 | Tuesday | Poster | |
| Abstract: A dynamic programming approach is used to optimize the tertiary structure of a protein. The input is the amino acid sequence. The algorithm builds the protein assembling the amino acids one by one, finding the minimum energy by trying a fixed number of rotations along the rotationally free bonds. The energy is calculated using a Molecular Mechanics model. The partial structure thus found is used to determine the next structure. This approach greatly reduces the complexity of the optimization. The results for sequences with a length of at most 100 amino acids show a remarkable similarity with experimentally determined structures. Increasing the number of rotations searched improves the resolution of the search and hence the accuracy of the prediction. Using a low resolution in the search allows the algorithm to produce a result in a few minutes, and the estimated structure still resembles the reference structure. This could allow the user to obtain a quick approximation to the desired structure. The software implementation allows the user to visually follow the assembly and optimization of the structure. | |||||
BibTeX:
@Poster{L100,
author = {Rosas-Trigueros Jorge L},
title = {Protein Structure Prediction using Dynamic Semi-Exhaustive Conformational Search},
booktitle = {Tuesday},
year = {100}
}
|
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| Rodriguez-Zavala Jose S. | Improvement of the Catalytic Efficiency of E. coli Lactaldehyde Dehydrogenase by Site Directed Mutagenesis | 101 | Monday | Poster | |
| Abstract: Aldehyde dehydrogenases (ALDHs) catalyze the conversion of aldehydes to their corresponding acids by the reduction of NAD(P)+. The physiologic role of ALDHs has been related to the detoxification of aldehydes from the organisms. However, some ALDHs participate in specific metabolic pathways. Phenylacetaldehyde dehydrogenase (PAD) and lactaldehyde dehydrogenase (ALD) from E. coli K12, are involved in the catabolism of L-phenylalanine and L-fucose, respectively. They share some structural and kinetic properties. One of the differences between these enzymes is that PAD uses NAD+ and NADP+, while ALD only uses NAD+. It has been proposed that E179 could be responsible for the exclusion of NADP+ from the coenzyme binding site. Nevertheless, E180 is present in both enzymes. Sequence analysis showed that in 10 residues around E180, only one amino acid was different between the two enzymes. This residue was T in PAD and F in ALD. Thus, we propose that T180 is exerting a steric effect impairing the binding of NADP+ to ALD. Mutation of F180 to T rendered an enzyme with the ability to use NADP+. Furthermore, ALD-F180T exhibited a 18-fold increment of Vm/Km with NAD+. The increase in catalytic efficiency was due to a diminution of Km of the enzyme for NAD+, accompanied by an increment in the Kd for NADH, which prompted a change in the rate limiting step of the reaction. In conclusion, residue F180 is exerting a steric effect on the coenzyme binding site, impairing not only the binding of NADP+, but also the correct binding of NAD+. | |||||
BibTeX:
@Poster{S.101,
author = {Rodriguez-Zavala Jose S.},
title = { Improvement of the Catalytic Efficiency of E. coli Lactaldehyde Dehydrogenase by Site Directed Mutagenesis},
booktitle = {Monday},
year = {101}
}
|
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| Rudiño-Pinera Enrique | The use of protein crystallography and NMR studies in elongated protein structures: the case of human fibronectin. | 102 | Tuesday | Poster | |
| Abstract: An important goal of structural studies of modular proteins is to determine the inter-module orientation, which often influences biological function. The N-terminal domain of human fibronectin (Fn) is composed of a string of five type 1 modules (F1). Despite their small size, to date F1 modules have proved intractable to X-ray structure solution, although there are several NMR structures available. Here, we present the first structures (two X-ray models and an NMR-derived model) of the (2)F1(3)F1 module pair, which forms part of the binding site for Fn-binding proteins from pathogenic bacteria. The crystallographic structure determination was aided by the novel technique of UV rayeartion damage-induced phasing. The individual module structures are very similar in all three models. In the NMR structure and one of the X-ray structures, a similar but smaller interdomain interface than that observed previously for (4)F1(5)F1 is seen. The other X-ray structure has a different interdomain orientation. This work underlines the benefits of combining X-ray and NMR data in the studies of multi-domain proteins. | |||||
BibTeX:
@Poster{Enrique102,
author = {Rudiño-Pinera Enrique},
title = {The use of protein crystallography and NMR studies in elongated protein structures: the case of human fibronectin.},
booktitle = {Tuesday},
year = {102}
}
|
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| Rojo Sandra E | Reconstruction of metabolic pathways in Streptomyces coelicolor: identification of missing genes in menaquinone biosynthesis | 103 | Monday | Poster | |
| Abstract: Genome-scale metabolic reconstructions (GSMR) emerge from the detailed analysis of genomic, physiological and biochemical data. These models are powerful tools for revealing pathway holes (Âmissing genesÂ) which represent enzymes needed in a metabolic pathway that cannot be connected to a specific gene using sequences similarities searches. Identification of missing genes after GSMR of model organisms, including Streptomyces coelicolor, have been reported. Several bioinformatics strategies can be used for searching and ranking candidate genes, including chromosomal clustering, co-occurrence and phylogenetic profiles. These in silico techniques, together with biochemical, genetic and post-genomic experimental data, have led to testable functional predictions. In the present work, we analyzed the menaquinone biosynthetic pathway, which consist of 8 enzyme functions (menABCDEFGH) in most organisms. Of these functions, the genome of S. coelicolor contains exclusively potential homologues of menA and menG, leaving the remaining enzymes of the pathway to be identified. This scenario is shared with some actinomycetes that are closely related to S. coelicolor, including members of the Salinispora and Frankia genera. Comparative genomic analyses, and biochemical considerations related to the synthesis of the aromatic ring of menaquinones, have led to the hypothesis that a polyketide synthase type-III conserved in these organism accounts for the lack of men genes. The experimental validation of the occurrence of a novel menaquinone biosynthetic pathway will be presented. This will include a proposal of the new biosynthetic pathway, in vivo mutagenesis experiments and metabolite profile analysis. Our results have a bearing in the evolution of enzymes and metabolic pathways. | |||||
BibTeX:
@Poster{E103,
author = {Rojo Sandra E},
title = {Reconstruction of metabolic pathways in Streptomyces coelicolor: identification of missing genes in menaquinone biosynthesis},
booktitle = {Monday},
year = {103}
}
|
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| Ruller Roberto | Thermostable Variants of the Recombinant xylanase a from Bacillus subtilis 1A1 Produced by Directed Evolution Show Reduced Heat Capacity Changes | 104 | Tuesday | Poster | |
| Abstract: Directed evolution techniques have been used to improve the thermal stability of the xylanase A from Bacillus subtilis (XylA). Two generations of random mutant libraries generated by error prone PCR coupled with a single generation of DNA shuffling produced a series of mutant proteins with increasing thermostability. The most Thermostable XylA variant from the third generation contained four mutations Q7H, G13R, S22P and S179C that showed an increase in melting temperature of 20oC. The thermodynamic properties of a representative subset of nine XylA variants showing a range of thermostabities were measured by thermal denaturation as monitored by the change in the far ultraviolet circular dichroism signal. Analysis of the data from these thermostable variants demonstrated a correlation between the decrease in the heat capacity change (deltaCP) with an increase in the midpoint of the transition temperature (Tm) on transition from the native to the unfolded state. This result could not be interpreted within the context of the changes in accessible surface area of the protein on transition from the native to unfolded states. Since all the mutations are located at the surface of the protein, these results suggest that an explanation of the decrease in deltaCP should include effects arising from the protein/solvent interface. | |||||
BibTeX:
@Poster{Roberto104,
author = {Ruller Roberto},
title = {Thermostable Variants of the Recombinant xylanase a from Bacillus subtilis 1A1 Produced by Directed Evolution Show Reduced Heat Capacity Changes},
booktitle = {Tuesday},
year = {104}
}
|
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| Roldan Maria Luisa | Interaction between beta-subunits of the Na+,K+-ATPase in vitro : Pull-Down assay | 105 | Monday | Poster | |
| Abstract: Previous work from our laboratory indicates that MDCK cells express the Na+, K+-ATPase at a given border provided the contacting cell expresses the dog β1-subunit as well. The cellÂcell interaction thus established would suffice to account for the polarized expression and positioning of the Na+,K+-ATPase in epithelial cells (Shoshani et al. 2005). Based on our findings, we postulated that two β-subunits of neighboring cells can interact directly and act as an adhesion molecule. To shed light on this hypothesis we are now studying protein-protein interactions between β-subunits of Na+,K+-ATPase. In the present work we tagged the dog β1-subunit with six Histidine residues (His6) at the cytoplasmic N-terminal end of the protein, created a stable clone of CHO cells that expresses it in the plasma membrane (CHO β1·His6) and utilized it to pull down another recombinant dog β1-subunit which lacks the intracellular and transmembrane domains of the protein and therefore is soluble (Secβ). Briefly, β1·His6 (extracted from CHO β1·His6 cells) was immobilized on beads coupled to Ni++ (Histrapp FF Amersham Biosciences) and incubated with soluble secβ. After excessive washing the β1·His6 was eluted with Imidazol (0.5M) and analyzed by Western blot. We found that Secβ was co-eluted with the His-tagged β-subunit suggesting that a specific β-β interaction occurred. Our future experiments are designed to elucidate whether this interaction is direct and if it involves the glycosilated moiety of the proteins. (Supported by CONACYT). | |||||
BibTeX:
@Poster{Luisa105,
author = {Roldan Maria Luisa},
title = { Interaction between beta-subunits of the Na+,K+-ATPase in vitro : Pull-Down assay},
booktitle = {Monday},
year = {105}
}
|
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| Salinas Juana del R | Bioinformatic analysis of herpes simplex virus type 2. | 106 | Tuesday | Poster | |
| Abstract: Membrane fusion is the strategy used by Herpes simplex virus type 2 (HSV-2) to infect cells. This event is meyearted by four viral glycoproteins gD, gB and gH-gL. Nowadays the complete process is unclear. Recent reports suggest that gH is HSV-2 fusion protein. The secondary structure of gH was determined by using several bioinformatic tools. Domain boundarie prediction methods showed that HSV-2 gH has three domains, one of them containing the previously reported fusion peptide and a heptad repeated region with high probability of forming coiled-coils, structure that has previously been associated to fusogenic properties in other viral fusion proteins. The other two domains are smaller and between them a previously reported antigenic site was found. One 3D model was obtained by using the I-tasser program, this model correlates in structure with the secondary structure determined previously. | |||||
BibTeX:
@Poster{R106,
author = {Salinas Juana del R},
title = {Bioinformatic analysis of herpes simplex virus type 2.},
booktitle = {Tuesday},
year = {106}
}
|
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| Rosas Cárdenas FF. | DIFFERENTIAL ANALYSIS OF PRICKLY PEAR (Opuntia spp) PROTEINS DURING RIPENING | 107 | Monday | Poster | |
| Abstract: Similar to gene expression profiling, proteomics offers the opportunity to examine simultaneous changes and to classify temporal patterns of protein accumulation occurring in complex developmental processes such as fruit ripening. It is possible to analyze protein function in fruit ripening through the characterization and identification of key molecules participating in the process. The aim of this work is to determine the differential expression of prickly pear proteins during ripening. The proteinic profiles were compared during the ripening process of three Opuntia morphospecies, with different ripening behavior: Naranjona, Blanca Cristalina, and Charola of early, early-intermeyearte, and intermeyearte-late ripening, respectively. Fruits were harvested at the green, green-ripe and ripe stages. The proteins were resolved on 2-DE gels with IPG strips (17 cm, pH 4-7, BioRad Readystrip, BioRad), stained with colloidal Coomassie and analyzed with Image master 2D Platinium 6.0 software (Amersham Biosciences). Specific proteins were recognized in each morphospecie which were differentially expressed during ripening. Constant and variable spots were excised, digested and analyzed by MALDI-TOF-MS and ESI-Q-TOF-MS/MS approaches. The analysis showed the highest differential expression pattern in the early ripening morphospecie. Differential proteins were found in the range pH 5.5 to 6.6 and with a MW of 50 to 100 KDa. A group of specific spots over-expressed in both, green and ripe stages, suggesting that these proteins maybe involved in different physiological processes. This behavior was similar to some other fruits, suggesting an active protein synthesis/degradation phenomena during fruit ripening. | |||||
BibTeX:
@Poster{FF.107,
author = {Rosas Cárdenas FF.},
title = { DIFFERENTIAL ANALYSIS OF PRICKLY PEAR (Opuntia spp) PROTEINS DURING RIPENING },
booktitle = {Monday},
year = {107}
}
|
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| Sanchez-Lopez Rosana | Transmission electron microscopy analysis of several light-chain amyloid-lke fibrils produced in vitro | 108 | Tuesday | Poster | |
| Abstract: Amyloidosis is a group of disorders characterized by the extracellular deposition of peptides and proteins as insoluble amyloid fibrils, which results in cell and organ dysfunction, and in many cases, to death. Amyloid fibrils structure Âcross beta-sheet- is formed by the assembly of intermolecularly hydrogen bonded beta-sheets that are assembled perpendicular to the fibril axis. It is well accepted that the primary structure of a given protein can bias it towards amyloid formation. Which specific sequence or structural determinats are required for amyloid formation remain, however, to be established. In order to gain insights into the structural organization of light-chain amyloid aggregation, we have performed a comparative analysis by negative staining and transmission electron microscopy of in vitro produced fibrils of recombinant-derived variable region (VL) protein fragments of lambda6 light chain, such 6aJL2 and its R25G variant (both designed and previously characterized in our group) as well as Jto1 and Wil1. In good agreement with data described by other authors, fibrils presented a yearmeter of ~10 nm, in average, and a variable length (more than 100 nm). We have observed distinctive morphological features associated to each fibrillogenic protein. 6aJL2 fibrils were mainly long and slender and formed by the lateral association of 2 or more rarely twisted protofibrils that, occasionally, joined to form bundles. On the other hand, the 6aJL2-R25G formed short and highly twisted fibrils. As described previously, Wil fibrils tended to be more twisted and shorter than those of Jto. 1 Wall JS et al (2004) J Mol Recognit 17(4): 323-31 This work was partially supported by grants from CONACyT (D44122Q) and DGAPA-UNAM (IN220707) |
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BibTeX:
@Poster{Rosana108,
author = {Sanchez-Lopez Rosana},
title = {Transmission electron microscopy analysis of several light-chain amyloid-lke fibrils produced in vitro},
booktitle = {Tuesday},
year = {108}
}
|
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| Le, EM Valenzuela Soto | Inhibition studies of porcine kidney betaine aldehyde dehydrogenase by oxidative stress | 109 | Monday | Poster | |
| Abstract: The enzyme betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) from mammals catalyzes the conversion of betaine aldehyde (BA) to glycine betaine (GB). GB is an organic osmolyte that contributes to the osmoregulation of renal cells. Mammal kidney normally is exposed to stress conditions due to its function. Oxidative stress is a physiological damage caused by reactive oxygen species (ROS) accumulation which can cause alterations to biomolecules.. To date any studies about changes produced in BADH by ROS has made. The goal of this study was to analyze BADH inhibition caused by one of the most stable ROS, hydrogen peroxide. Inhibition assays were carried out at different peroxide concentrations (0.01 to 0.2 mM) at saturant (0.5 mM NAD+ and BA) and subsaturant (0.05 NAD+ and BA) substrate concentration. The kinetic constants of BADH without inhibitor (Vmax = 2.9 ± 0.10 µmol min-1 mg-1, KmNAD = 58.4 ± 7.5 µM, KmBA = 109 ± 11 µM) were modified in presence of hydrogen peroxide. Inhibition analysis show mixed inhibition for NAD (Vmax = 1.72 ± 0.03, Km = 37.8 ± 3 µm, Ki = 115 ± 28 µM) and no competitive for BA (Vmax = 1.72 ± 0.03, Km = 91.8 ± 6 µm, Ki = 400 ± 49 µM). Even when the affinity of BADH for its substrates increases, its efficiency decreases. Our results suggest that the inhibition caused to BADH, compromise the synthesis of the GB osmolyte, increasing the risk of renal injury. | |||||
BibTeX:
@Poster{,
author = {J. A. Rosas Rodríguez},
title = {Inhibition studies of porcine kidney betaine aldehyde dehydrogenase by oxidative stress},
booktitle = {Monday},
year = {109}
}
|
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| Tellez Luis A. | The Role of Dimerization in the Structure of TIM: a Molecular Dynamics Approach | 110 | Tuesday | Poster | |
| Abstract: Triosephosphate isomerase (TIM) is always present as an oligomer formed by identical (beta/alpha)8 barrels (a homodimer in mesophiles and a homotetramer in some hyperthermophiles). Although each TIM monomer contains its own active site and there is no evidence of allostery or cooperativity between subunits, only the oligomeric forms are functional. Thermodynamically, TIM dimerization contributes to most of the free energy of folding. Thus, TIM function and stability are intimately linked. To gain insight into the role of oligomerization in the stability and function of TIM we employed an in silico approach. We used molecular dynamics (MD) to dissect the relative contribution of oligomerization to the stability of the monomers and to the integrity of the catalytic site. We performed MD simulations of TIM dimers and monomers from two different species: Entamoeba histolytica (1M6J) and Saccharomyces cerevisiae (1YPI). All simulations were performed using the GROMACS suite and the GROMOS96 53a6 force field. Several global and local structural features were analyzed from 10 nanosecond trajectories carried out at 300 and 500 K. The results obtained show that the behavior of TIMs from both species is quite similar. A clear relationship between the oligomeric state and global stability was not found: changes in radius of gyration, integrity of the beta-sheet core and loss of secondary structure are comparable among free and associated monomers. Nevertheless, the geometry of the catalytic site proved to be extremely sensitive to monomerization. Taken together, these results support the hypothesis that dimerization optimizes active site geometry. | |||||
BibTeX:
@Poster{A.110,
author = {Tellez Luis A.},
title = {The Role of Dimerization in the Structure of TIM: a Molecular Dynamics Approach},
booktitle = {Tuesday},
year = {110}
}
|
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| Rudiño-Pinera Enique | Vibrational modifications produced by single residue modifications in Glucosamine 6-P deaminase. | 111 | Monday | Poster | |
| Abstract: E. coli GlcN6P deaminase catalyses the isomerisation and deamidation of glucosamine 6-P (GlcN6P), yielding fructose 6-P (Fru6P) and ammonia. This enzyme is allosterically activated by the N-acetyl derivative of the substrate, GlcNAc6P; it displays intense homotropic cooperativity, which turns to hyperbolic binding or kinetics at saturating activator concentration (K-system). The allosteric properties of GlcN6P deaminase can be accurately described by the MWC model. T. A wide set of mutations was constructed to explore its allosteric function. Some mutations reverted the effect of the activator, which became inhibitor above a critical GlcN6P concentration. Substrate inhibition was also apparent, which was abolished by the allosteric effector. In this work the correlation between the crystallographic data of several mutant enzymes and its kinetical behaviour will be explored. | |||||
BibTeX:
@Poster{Enique111,
author = {Rudiño-Pinera Enique},
title = {Vibrational modifications produced by single residue modifications in Glucosamine 6-P deaminase.},
booktitle = {Monday},
year = {111}
}
|
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| Toledo-Nunez Citlali | Residual structure in Laccase from Myceliophthora thermophyla after thermal unfolding | 112 | Tuesday | Poster | |
| Abstract: Recently, it has been proposed that the residual secondary structure present in a thermally unfolded protein may be related to its thermostability. Comparative studies carried out with homologous proteins obtained from mesophilic and thermophilic organisms [Hollien and Marqusee, 1999] have found smaller denaturation heat capacity changes DCp for the thermophilic protein in comparison to its mesophilic homologue. Furthermore, such a small DCp is thought to be a reflection of the presence of residual structure in the thermally unfolded macromolecule. A recent study of the thermophilic laccase from Myceliophthora thermophyla, (MtL) [Lopez-Cruz et al., 2006] revealed that this monomeric enzyme denatures with a very small enthalpy change (DHU) of only 1.8 cal/g. In comparison, the value of DHU for egg-white lysozyme is 10.8 cal/g. This report has led us to think that small DCp and DHU values are both indicative of the presence of residual structure. Therefore, we are doing a detailed study of the thermal denaturation of MtL and another laccase from a mesophile fungus. For these studies we have done a biochemical characterization of the enzyme, and recorded CD spectra at different values of pH. Results suggest that at neutral pH some secondary structure is conserved after thermal unfolding (up to 90 ºC); at low pH values, however, CD spectra gradually become similar to that of a typical, thermally unfolded protein, such as lysozyme. Lowering of the pH also causes a decrease in the temperature at which MtL is half denaturated. |
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BibTeX:
@Poster{Citlali112,
author = {Toledo-Nunez Citlali},
title = {Residual structure in Laccase from Myceliophthora thermophyla after thermal unfolding},
booktitle = {Tuesday},
year = {112}
}
|
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| Ruiz-Granados Y. G. | Oligomeric States of the Yeast Plasma Membrane H(+)-ATPase. Enzyme Kinetics and Intrinsic Fluorescence Study. | 113 | Monday | Poster | |
| Abstract: The yeast plasma membrane H+-ATPase is a member of the P-type ATPase family (Serrano et al, 1986). The H+-ATPase pumps protons out of the cell generating an inward ion gradient which is used for nutrient transport (Serrano et al, 1986). Detailed structural studies for the plasma membrane H+-ATPase from yeast and other sources are scarce (Morsomme et al 2000). However, the native state of the H+-ATPase is controversial as several oligomeric states (decamer, octamer, hexamer, dimer and including the monomer) have been proposed (Morsomme et al, 2000; Cyrklaff et al, 1995; Chadwick et al, 1987; Goormaghtigh et al, 1986; Bowman et al, 1985). The plasma membrane H+-ATPase from Kluyveromyces lactis was fully purified after centrifugation on a trehalose concentration gradient. However, H+-ATPase was in a highly aggregated state as tested by light scattering. The protein aggregates were separated by size exclusion chromatography on Superose 6. The elution profile exhibited two main absorbing peaks at 280 nm. Peak I consisted solely of the ATPase in a hexameric state (600 kDa) as revealed by means of blue native electrophoresis, while peak II was a mixture of different oligomeric states; hexamers (600 kDa), tetramers (400 kDa), dimers (200 kDa) and monomers (100 kDa) were observed. ATP saturation kinetics for both peaks, showed a sigmoid (cooperative) dependence; Hill number (n) was slightly higher for the hexamer (n= 1.97 ± 0.21) than for the mixture of oligomeric states. While S0.5 values were similar; S0.5= 330 ± 20 μM ATP. Interestingly, the maximum velocity for the hexamer was three times higher (Vmax= 17.68 ± 0.67 μmols ATP min-1mg prot-1) than for the mixture of oligomeric states (Vmax= 5.19 ± 0.25 μmols ATP min-1.mg prot-1). Intrinsic fluorescence studies were also performed; fluorescence intensity for the hexamer was three times higher than fluorescence for the mixture of oligomeric states. Further, H+-ATPase fluorescence was quenched upon nucleotide binding and showed a hyperbolic dependence on nucleotide concentration. The maximum fluorescence quenching was observed in the presence of AMP-PCP; an ATP analogue. The hexameric state displayed the highest affinity for nucleotides; dissociation constant (Kd) for ATP and ADP were 120 ± 3 and 200 ± 6 μM respectively. Based in the present results, the hexamer is proposed to be the fully activated state for the H+-ATPase, as it displayed both Vmax and intrinsic fluorescence three times higher than the oligomers mixture. The other oligomers detected (namely dimers and tetramers) would represent probably partially activated states in way of hexamer formation. This is in accord with the idea that, during activation by glucose, phosphorylation of Thr912 liberates the carboxyl-terminus from its auto-inhibitory position (Lecchi et al, 2005) allowing the carboxyl-terminus of a monomer to interact with the next monomer to stabilize the hexamer (Kulbrandt et al, 2002, 2004). REFERENCES Â Serrano, R., Kielland-Brandt, M.C. and Fink, G.R. (1986) Nature, 319:689-693. Â Morsomme, P., Slayman, C.W. and Goffeau, A. (2000) Biochim. Biophys. Acta,1469:133-157. Â Cyrklaff, M., Auer, M., Kuhlbrandt, W. and Scarborough, G. A. (1995) EMBO J., 14:1854-1857. Â Chadwick, C.C., Goormaghtigh, E. and Scarborough, G.A. (1987) Arch. Biochem. Biophys., 252:348-356. Â Goormaghtigh, E., Chadwick, C. and Scarborough, G.A. (1986) J. Biol. Chem., 261:7466-7471. Â Bowman, B.J., Berenski, C.J. and Jung, C.Y. (1985) J. Biol. Chem., 260:8726-8730. Â Lecchi, S., Allen, K. E., Pardo, J. P., Mason, A. B. and Slayman, C. W. (2005) Biochemistry, 44, 16624-16632 Â Kühlbrandt, W., Zeelen, J., and Dietrich, J. (2002) Science 297, 1692-1696. | |||||
BibTeX:
@Poster{G.113,
author = {Ruiz-Granados Y. G.},
title = { Oligomeric States of the Yeast Plasma Membrane H(+)-ATPase. Enzyme Kinetics and Intrinsic Fluorescence Study.},
booktitle = {Monday},
year = {113}
}
|
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| Torres Marilu | A study of mutants in tyrosines 102 and 103 of triosephosphate isomerase from Trypanosoma cruzi | 114 | Tuesday | Poster | |
| Abstract: Triosephosphate isomerase (TIM) is a glycolytic enzyme formed by two identical monomers that has catalytic activity only as a dimer. The interface between the monomers is a better target for species-specific drug design than the active site, because the amino acids in that first region are less conserved. Searching for binding sites for organic molecules on the protein, in previous work, we crystallized TIM from Trypanosoma cruzi (TcTIM) in the presence of hexane. Three hexane molecules bound to the TcTIM crystal, two of which were at the interface. In later studies, we solved the structure of a crystal of TcTIM in the presence of a molecule that selectively inhibited TcTIM: (3-(2-benzothiazo-lylthio)-1-propanesulfonic acid or compound 8. The single molecule of compound 8 that bound to TcTIM was very close to the binding site of the two interface hexanes, and at a distance of less than 4 Å of some amino acids of the interface, namely Phe 75, Tyr 102 and Tyr 103. These residues belong to a group of aromatic amino acids that form a hydrophobic cluster in the interface of TcTIM. In order to determine the importance of these residues for the binding of several inhibitors of TcTIM we have studied, we mutated Tyr 102 and 103 in TcTIM and generated mutants Y102V, Y103V, Y102V/Y103F and Y102F/Y103F. We determined the kinetic constants of these proteins and found that there are no significant differences between the wild type and the mutant enzymes. Nevertheless, mutants Y102V and Y103V are less stable to dilution and showed increased susceptibility to compound 8 and to 6,6bisbenzothiazole-2,2diamine (compound V7) when compared to wild type TcTIM. Mutants Y102V/Y103F and Y102F/Y103F are stable and are less susceptible to these compounds. |
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BibTeX:
@Poster{Marilu114,
author = {Torres Marilu},
title = {A study of mutants in tyrosines 102 and 103 of triosephosphate isomerase from Trypanosoma cruzi},
booktitle = {Tuesday},
year = {114}
}
|
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| Salum Livia B | Library Design, Ligand Docking and Structure-Based Virtual Screening in the Lead Discovery of Novel Estrogen Receptor Modulators | 115 | Monday | Poster | |
| Abstract: The Estrogen Receptor (ER) is an attractive drug target for breast cancer therapy. As part of our efforts to both understand the molecular basis of drug interaction and identify novel promising ER modulators, we have created a large small-molecule chemical library which was used for ligand docking and structure-based virtual screening studies. In this process, about 200,000 compounds were selected from the free database ZINC (the default version 2006 ZINC) and subjected to in-house molecular and ADME property filters in order to reduce the chemical space according to features presented by a series of 50 structurally distinct ER ligands. The X-ray crystal structure of ER in complex with an antagonist was collected from the Protein Data Bank (PDB code: 1XP1), analyzed and prepared for ligand docking and structure-based studies. Molecular docking and scoring were performed using the program GOLD 3.0. A final subset of 20 ligand candidates was then selected for molecular modeling studies involving the predicted binding modes and intermolecular interactions related to biological activity. | |||||
BibTeX:
@Poster{B115,
author = {Salum Livia B},
title = {Library Design, Ligand Docking and Structure-Based Virtual Screening in the Lead Discovery of Novel Estrogen Receptor Modulators},
booktitle = {Monday},
year = {115}
}
|
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| Vazquez-Duhalt Rafael | Rational molecular design of a peroxidase from cytochrome | 116 | Tuesday | Poster | |
| Abstract: The industrial application of enzymes is growing and peroxidases have interesting potential applications in many different fields. However, present and future commercial uses have been limited, mainly, by the low stability of peroxidases in the presence of their natural substrate, hydrogen peroxide. Cytochrome c is also able to catalyze peroxidase-like reactions. During the oxidative self-inactivation of cytochrome c, four molecular events were detected and kinetically characterized; heme destruction, loss of the iron-sulfur coordination, protein multimerization, and protein inactivation. Electron transfer balance from the four molecular events account for 100% of the electrons transferred in the first minute of protein incubation with hydrogen peroxide. The oxidative stabilization of iso-1-cytochrome c, was achieved through the rational alteration of the intra-molecular electron-transfer network. Our results demonstrated that catalysis by hemeproteins as peroxidases is a naturally imperfect process. In the presence of exogenous substrates, the porphyrin ring and the protein backbone act as simultaneous and competing electron sources, as was demonstrated by the iso-1-cytochrome c stabilization when lower redox potential exogenous substrates were used. Protein destruction seems then to arise as a consequence of unproductive electron abstraction pathways, whereas the favorable partition of the oxidative equivalents towards the added substrate results in a protective effect. A redox-based design strategy was followed until an iso-1-cytochrome c variant, fully stable at catalytic concentrations of hydrogen peroxide, was obtained. The results presented here clearly demonstrate that the rational modulation of the intra-molecular electron transfer network is a useful tool to prevent suicide inactivation of heme-peroxidases. | |||||
BibTeX:
@Poster{Rafael116,
author = {Vazquez-Duhalt Rafael},
title = {Rational molecular design of a peroxidase from cytochrome},
booktitle = {Tuesday},
year = {116}
}
|
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| Sandoval Mario | Reconstruction of metabolic pathways in Streptomyces coelicolor: identification of missing genes in cardiolipin biosynthesis | 117 | Monday | Poster | |
| Abstract: Genome-scale metabolic reconstructions (GSMR) emerge from the detailed analysis of genomic, physiological and biochemical data. These models are powerful tools for revealing pathway holes (Âmissing genesÂ) which represent enzymes needed in a metabolic pathway that cannot be connected to a specific gene after sequence similarity searches. Identification of missing genes after GSMR of model organisms, including Streptomyces coelicolor, have been reported. Several bioinformatics strategies can be used for searching and ranking candidate genes. These in silico techniques, together with biochemical, genetic and post-genomic experimental data, have led to testable functional predictions. In the present work, we discuss an example of the application of these integrative analyses in finding the cardiolipin synthase (CLS) gene in S. coelicolor, previously identified as missing after GSMR, which is consistent with the fact that cardiolipin could be detected in membrane extracts of this organism. Since two forms of CLS, i.e. the prokaryote type, which proceeds via condensation of two molecules of phosphatidyl glycerol, and a eukaryotic form that transfers one phosphatidyl moiety from CDP-yearcyl glycerol to phosphatidyl glycerol, have been recognized, we hypothesized that latter CLS could be present in this organism. Indeed, a potential eukarytic-like CLS was detected using genome context and phylogenetic analyses, which was cloned and over-expressed to look at its CLS activity. A S. coelicolor mutant and another CL deficient strain were then used for in vivo complementation assays. Our results suggest that this enzyme may represent a novel drug target present in certain pathogenic actinomycetes, such as Mycobacterium. | |||||
BibTeX:
@Poster{Mario117,
author = {Sandoval Mario},
title = {Reconstruction of metabolic pathways in Streptomyces coelicolor: identification of missing genes in cardiolipin biosynthesis},
booktitle = {Monday},
year = {117}
}
|
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| Vazquez-Limon Consuelo | Saturation mutagenesis at position 38 from LDH of Bacillus subtilis and its effect on growth rate | 118 | Tuesday | Poster | |
| Abstract: It has been reported (1) that substitution of the conserved aspartate residue by glycine at position 52 in LDH from B. stearothermophilus (which correspond to aspartate 38 in LDH from B. subtilis) causes a significant decrement on the NADH affinity and also a decrement on kcat, resulting in a two orders of magnitude decreased kcat /Km. We constructed the equivalent D38G on LDH from B. subtilis (which shares 67% identity with LDH from B. stearothermophilus). As expected, mutant D38G yields very small colony forming strains under anaerobic culture conditions. A library was constructed by saturation mutagenesis at position G38 and selected on E. coli VAL23, under anaerobic conditions, on LB meyear supplemented with Na2CO3 (as buffer) and glucose. Growth rate (measured as colony size) and consequently an improved NAD+ regeneration rate was the screening criterion for mutants later identified by nucleotide sequence as G38A, G38C, G38E and G38D. It is important to note that under aerobic culture conditions clones containing most of the 20 amino acid substitutions were found; however, most of them were unable to form easily visualized colonies when grown on anaerobic culture conditions. Specific growth rate on liquid anaerobic meyear of E. coli VAL23 transformed with variants D38G (0.15 h-1), G38A (0.41 h-1), G38C (0.51 h-1) and wild type LDH (0.59 h-1) show that the differences in colony yearmeter correlates with specific growth rate. Besides, the specific growth rate of each clone could be directly associated with the rate of the limiting enzymatic step in the strain, as the specific activity of the purified enzymes was found as D38G (6.1 E04), G38A (1.1 E05), G38C (2.0 E05) and wild type LDH (6.4 E05). References 1. Holmberg N, Ryde U, Bülow L (1999) Redesign of the coenzyme specificity in L-lactate dehydrogenase from Bacillus stearothermophilus using site-directed mutagenesis and meyear engineering. Protein eng. 12: 851-856. | |||||
BibTeX:
@Poster{Consuelo118,
author = {Vazquez-Limon Consuelo},
title = {Saturation mutagenesis at position 38 from LDH of Bacillus subtilis and its effect on growth rate},
booktitle = {Tuesday},
year = {118}
}
|
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| Sarai Teloxa | Spectroscopic characterization of the Cu(II) coordination sites in the 96-115 region of the human prion ptotein | 119 | Monday | Poster | |
| Abstract: The misfolding of the prion protein (PrP) into a β-sheet conformation is associated with Transmissible Spongiform Encephalopathies, like the mad cow disease, and kuru and Ceutzfeld-Jackob disease in humans. Human PrP is capable of binding up to six copper ions, four of which bind in the octarepeat region, while a fifth and sixth binding sites have been identified in the flexible region between residues 90 and 126. This region plays a key role in the misfolding of PrPC (PrP Cellular) and Cu2+ binding to His 96 and His 111 is thought to promote the conformational change. In this study, the nature of the Cu2+ coordination sites in the 92-115 regions are characterized using different synthetic peptides and spectroscopic tools like UV-VIS absorption, circular dichroism and electron paramagnetic resonance. Further insight into the electronic structure of these sites is gained through pH studies and spectroscopic analysis. In particular, the electronic structure of the Cu(II) complex with the peptide fragment 92-96 (GGGTH) is described by DFT calculations. | |||||
BibTeX:
@Poster{Teloxa119,
author = {Sarai Teloxa},
title = { Spectroscopic characterization of the Cu(II) coordination sites in the 96-115 region of the human prion ptotein},
booktitle = {Monday},
year = {119}
}
|
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| Vega Vanessa | Site directed mutagenesis and over-expression of Neurospora crassa catalase/peroxidase | 120 | Tuesday | Poster | |
| Abstract: Catalases, peroxidases and catalase/peroxidases (CPs) are involved in defense mechanisms against reactive oxygen species. CPs have a predominant catalase activity but, different to catalases, they exhibit substantial peroxidatic activity. CPs are present only in bacteria and fungi; fungal enzymes probably arose by lateral transfer of a bacterial gene. Neurospora crassa CAT-2 shows ~60% identity with bacterial CPs but has an extra loop of 25-30 amino acid residues. CAT-2 is a cytosolic enzyme that is induced during late stationary growth, asexual spore formation, growth on poor carbon sources, and under heat shock. A cat-2 null mutant strain produced more coniyear during the late stationary growth, and even more when on a poor carbon source. Because of CP bi-functionality, elucidation of which activity is related to this phenotype is necessary. Using site directed mutagenesis, we obtained a W90A substitution of CAT-2. A bacterial enzyme with the equivalent substitution presents only peroxidase activity. cat-2 null mutant strain was transformed with the W90A mutated gene. Contrary to what was expected, the enzyme produced lacked both catalase and peroxidase activity even when the transformant was grown on a poor carbon source. To elucidate how Neurospora CP differs from the bacterial enzymes, a 6 histidine tagged cat-2 was expressed in Escherichia coli and the enzyme was purified by nickel-affinity chromatography. We will test several mutations of cat-2 to obtain a mono-functional enzyme and transform Neurospora with these genes. | |||||
BibTeX:
@Poster{Vanessa120,
author = {Vega Vanessa},
title = {Site directed mutagenesis and over-expression of Neurospora crassa catalase/peroxidase},
booktitle = {Tuesday},
year = {120}
}
|
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| Serratos Álvarez Iris N. | Assessing electrostatic interactions in enzyme-inhibitor complexes: Triosephosphate isomerase with 2-phosphoglycolate | 121 | Monday | Poster | |
| Abstract: One fundamental method for the study of electrostatic effects in molecular recognition is the resolution of the Poisson-Boltzmann (PB) equation for the system in question. For this purpose we use the structure of the molecular couple at atomic resolution considering a determined dielectric constant for the complex, whereas the solvent is treated implicitly as a continuum dielectric medium. The solution is obtained through numerical methods that allow obtaining the electrostatic potential generated by the distribution of charges and dielectrics in a determined ionic strength. In this work, the binding energies (ΔGb) of triosephosphate isomerase from Saccharomyces cerevisiae, ScTIM, with the inhibitor 2-phosphoglycolate (2PG), determined by fluorimetric titrations at different ionic strengths (0 - 1.0 M NaCl) at pH 7.4 are reported. These ΔGb values are compared with the sum of calculated energies as electrostatic and nonpolar contributions. For the electrostatic part the APBS programs (Adaptive Solver Poisson-Boltzmann) were employed by using the crystalographic structure of the ScTIM-2PG complex. On the other hand, the nonpolar contribution was evaluated as the released energy by the decrease in the cavity area of solvent when the complex is formed, such energy being proportional to the change of solvent accessible surface area (ΔASA), considering a surface tension of 0.021 kJ mol-1Ǻ-2 for water. Close to physiological conditions (pH 7.4, 25 ºC and ionic strength of 0.25) the calculated value of ΔGb was of -20.2 kJ mol-1 (ΔGb,elec = -12.9 kJ mol-1 and ΔGb, nonpolar = -7.38 kJ mol-1), which compares well with the experimental value of -22.3 kJ mol-1. | |||||
BibTeX:
@Poster{N.121,
author = {Serratos Álvarez Iris N.},
title = { Assessing electrostatic interactions in enzyme-inhibitor complexes: Triosephosphate isomerase with 2-phosphoglycolate},
booktitle = {Monday},
year = {121}
}
|
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| Vega-Badillo Joel | Directed evolution of LDH of Bacillus subtilis to improve L-lactate production in E.coli. | 122 | Tuesday | Poster | |
| Abstract: LDH from diverse organisms causes a cell toxicity effect when over-expressed in E. coli. In this work, we found a negative effect on bacterial growth when E.coli VAL23 transformed with pTrclctE was grown even at low levels of IPTG inducer (0.05 mM). To remove the toxicity effect on bacterial growth, an LDH mutant library was generated by error-prone PCR and selected on anaerobic meyear in the presence of IPTG. Variant 6T3 included six amino acid substitutions (N3D, A22T, D141E, A145V, K230R and Y235F), and variant 2P included four substitutions (K87N, L120I, D270E, L311F). Substitutions were mapped on the known quaternary structure of LDH of B. stearothermophilus (1) and found on the solvent exposed protein surface, far from subunit interfaces and far from ligand binding sites. When cultured on microaerobic, pH controlled liquid cultures (M9 meyear, 40 g glucose/l and 0.1 mM IPTG), a rapid lactate production was observed with the bacteria transformed with variants 6T3 and 2P resulting from higher specific growth rates as compared to bacteria transformed with the wild type clone. References 1.- Schar HP, Zuber H, Rossman MG (1982) Crystallization of lactate dehydrogenase from Bacillus stearothermophilus. J. Mol. Biol. 154: 349-353. |
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BibTeX:
@Poster{Joel122,
author = {Vega-Badillo Joel},
title = {Directed evolution of LDH of Bacillus subtilis to improve L-lactate production in E.coli.},
booktitle = {Tuesday},
year = {122}
}
|
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| Solis Calero Christian | Homology modeling of L-amino acid oxidase from Bothrops jararacussu and docking of its reversible inhibitors | 123 | Monday | Poster | |
| Abstract: Bothrops is a genus of venomous pit vipers found in Central and South America, responsible for more human deaths in the Americas than any other group of venomous snakes. For that reason, it is important to develop agents that neutralize activities of their components. One of the components is l-amino acid oxidase (LAO, EC 1.4.3.2), a flavoenzyme, which is responsible for the oxidative deamination of l-amino acids. Elucidation of the x-ray crystallographic structure of LAOs from another snakes and disposal of amino acid sequence from Bothrops jararacussu has opened the way for molecular modeling studies. In this research homology modeling of the LAO from B. jararacussu was performed based on the crystal structures determined for another LAOs (PDB codes 1TDO and 2IID) by using the software MODELLER 9v1. With the aid of the molecular mechanics, the final model was obtained and assessed subsequently by PROCHECK. With this model, a docking study with substrates and 6 reversible inhibitors was performed using AUTODOCK 4. The result of these studies generated thermodynamic properties, such as inhibition constants (Ki) for substrates and inhibitors. Moreover, 3D pictures of inhibitor-enzyme complexes afforded valuable data regarding the binding orientation of each inhibitor in the active site of LAO, giving important information to develop better inhibitors to probe experimentally. | |||||
BibTeX:
@Poster{Christian123,
author = {Solis Calero Christian},
title = {Homology modeling of L-amino acid oxidase from Bothrops jararacussu and docking of its reversible inhibitors},
booktitle = {Monday},
year = {123}
}
|
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| Wright Helena | Structural studies of the dual-substrate TIM-barrel phosphoribosyl isomerase A (PriA) from Streptomyces coelicolor | 124 | Tuesday | Poster | |
| Abstract: Phosphoribosyl isomerase A (PriA) is a dual-substrate (beta alpha)8-barrel, involved in both histidine (HisA) and tryptophan (TrpF) biosynthesis [1]. Despite the availability of a previously obtained 3D structure of PriA [2], the structure / function relationship remains poorly defined. Since PriA has ancient-like features, i.e. a dual-substrate specificity within a physiological context [1], this TIM-barrel represents a suitable model system to provide valuable insights for enzyme evolution. A complete crystallographic structure of PriA from Streptomyces coelicolor has been resolved at 1.8 Å using Multiwavelength Anomalous Dispersion (MAD) together with crystals substituted with seleno-methionine. The complete crystallographic structure of PriA reported here has revealed well defined functional loops 1 and 6 (beta®alpha), that appeared disordered in the previously available structure [2]. The loops interact via hydrogen bonding which closes the active site towards the C-terminal end of the barrel. An unusual contact involving a residue in loop 6 has also been detected. Site-directed mutagenesis and in vivo functional analysis has allowed the identification of residues involved in substrate specificity and catalysis. The information provided from the new crystallographic model of PriA has revealed a proposed Âclosed conformer of PriA. [1] Barona-Gómez, F., Hodgson, D.A., EMBO Rep 2003, 4: 296-300. [2] Küper, J., Doenges, C., Wilmanns, M., EMBO Rep 2005, 6: 134-139. |
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BibTeX:
@Poster{Helena124,
author = {Wright Helena},
title = {Structural studies of the dual-substrate TIM-barrel phosphoribosyl isomerase A (PriA) from Streptomyces coelicolor},
booktitle = {Tuesday},
year = {124}
}
|
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| Sotelo-Mundo Rogerio R. | Structural basis for the increased substrate affinity in marine invertebrate lysozyme | 125 | Monday | Poster | |
| Abstract: Lysozymes are components of the innate immune system, functioning as antibacterial proteins, and in some cases as digestive enzymes. The lysozyme from a marine shrimp Litopenaeus vannamei, has a lytic activity against Gram (+) and Gram (-) bacteria. The shrimp lysozyme is one of the largest type-C lysozymes reported to date. Its sequence contains a seven-residue insertion and a nine-residue extension at the C-terminus, only found in penaeid crustaceans. The enzyme was overexpressed by recombinant methods. In general, it was observed that this protein tends to form oligomers in solution and was found to have a binding constant an order of magnitude larger compared to the hen and duck egg-white lysozymes using chitotriose as ligand. The binding reaction was exothermic. Besides, a conformational change measured by circular dichroism occurred upon ligand binding, and led to an increase in the b-sheet structure content. The additional sequences present in shrimp lysozyme may contribute to its high catalytic activity and binding affinity for chitotriose, and explain the differences in its secondary structure content. The presence of hydrophobic residues at C-terminus can promote the activity on Gram (-) bacteria. The crystal structure of the shrimp lysozyme solved with and without ligand, revealed a large active site cavity and a net charge that favors catalysis and binding. These results reveal that marine invertebrates evolved to have a highly antibacterial lysozyme that is a key component of the innate defense system. Supported by grants 48991-Z;36928-B from CONACYT (Consejo Nacional de Ciencia y Tecnologia). |
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BibTeX:
@Poster{R.125,
author = {Sotelo-Mundo Rogerio R.},
title = { Structural basis for the increased substrate affinity in marine invertebrate lysozyme},
booktitle = {Monday},
year = {125}
}
|
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| Zaldivar-Machorro Victor | Structural Effects of the Chemical Modification of the Betaine Aldehyde Dehydrogenase from Pseudomonas aeruginosa by Disulfiram and its Metabolites | 126 | Tuesday | Poster | |
| Abstract: In the pathogen Pseudomonas aeruginosa the reaction catayzed by betaine aldehyde dehydrogenase (PaBADH) appears to play an important role during infection, suggesting that this enzyme may be a suitable drug target. Moreover, PaBADH inhibition would lead to the build-up of the toxic betaine aldehyde. Disulfiram (DSF), a drug used for years in treatment of alcoholism, inactivated this enzyme in a time- and dose-dependent manner, and also produced enzyme aggregation. Since DSF is readily metabolised in vivo to methyl diethylthiocarbamoyl sulfoxide (MeDTC-SO) and sulfone (MeDTC-SO2), and to diethyldithiocarbamoyl sulfoxide (MeDDTC-SO) and sulfone (MeDDTC-SO2), we tested these compounds in vitro as PaBADH inhibitors, as well as investigated their effect on PaBADH structure by turbidometry, gel filtration chromatography, intrinsic and extrinsic protein fluorescence, and circular dichroism. Chemical modification of PaBADH by DSF metabolites did not affect the quaternary structure of the enzyme, but it did have significant effects on tertiary and secondary structures, as well as on the enzyme thermostability. MeDTC-SO2 , which is the most potent PaBADH inhibitor among the DSF metabolites, increased the apparent Tm of PaBADH 15 oC, an opposite effect to that of DSF. These differences are most likely related to the different modification of the catalytic cysteine residue: DSF foms a bulky mixed disulfide whereas DSF metabolites form smaller carbamoyl-cysteine adducts. The polarity of the introduced group might also be important. Our results clearly show how the modification of enzyme catalytic residues may have not only functional but also structural consequences. Supported by DGAPA-UNAM grant IN206505 | |||||
BibTeX:
@Poster{Victor126,
author = {Zaldivar-Machorro Victor},
title = {Structural Effects of the Chemical Modification of the Betaine Aldehyde Dehydrogenase from Pseudomonas aeruginosa by Disulfiram and its Metabolites},
booktitle = {Tuesday},
year = {126}
}
|
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| Souza Diorge P | Characterization of the VirB7-VirB9 interaction and initial NMR assignment of VirB7 from Xanthomonas Type IV Secretion System | 127 | Monday | Poster | |
| Abstract: Xanthomonas axonopodis pv. citri (Xac) is a phytopathogen that causes citrus canker in orange trees. Among the possible virulence determinants in the Xac genome is the chromosomal Type IV secretion system (T4SS), an important transenvelope apparatus that secrets proteins and DNA in many plant and animal pathogens. The model T4SS found in Agrobacterium tumefaciens is constituted by twelve structural proteins: VirB1-VirB11 and VirD4. We expressed specific Xac VirB7 (with a His-tag) and VirB9 fragments in Escherichia coli. The proteins were purified by standard chromatographic methods and characterized by SDS-PAGE, mass spectrometry, fluorescence and circular dichroism. VirB9 was expressed as an insoluble polypeptide, purified in the presence of urea and refolded by yearlysis. The VirB7-VirB9 interaction was assayed by fluorescence titration, indicating a Kd of ~4x10-8M. For NMR studies, VirB7 was induced in minimum meyear for incorporation of 15N and 13C and the His-tag was removed by thrombin proteolysis. Several multidimensional spectra were collected for resonance assignment, including 15N-HSQC, 13C-HSQC, HNCO, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, HBHA(CO)NH, 15NTocsy-HSQC and HC(CO)NH-Tocsy. More than 90% of the backbone and the side chain Cβ and Hβ have been assigned. The VirB7-VirB9 interaction was monitored by 15NHSQC of 15N-labelled VirB7 before and after addition of VirB9, indicating that the VirB7 N- and C-terminal remain highly flexible in the VirB7-VirB9 complex. Supported by: LNLS, CNPq and Fapesp |
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BibTeX:
@Poster{P127,
author = {Souza Diorge P},
title = {Characterization of the VirB7-VirB9 interaction and initial NMR assignment of VirB7 from Xanthomonas Type IV Secretion System},
booktitle = {Monday},
year = {127}
}
|
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| Inés Burgos | A novel theoretical tool for the analysis of calorimetric data of oligomeric proteins | 128 | Tuesday | Poster | |
| Abstract: The thermodynamic parameters for the process of protein unfolding can be obtained through differential scanning calorimetry. However, the unfolding process may not be a two state one. Between the native and the unfolded state there may be association or dissociation processes or the formation of an intermediate state. As a consequence of this, the interpretation of the calorimetric data can not be straightforward. In this work we present two general models for the unfolding process of an oligomeric protein in which an intermediate state, or is present: Model I Model II In model I, the first step represents the dissociation of the oligomer into the monomeric native species and the second step represents the denaturation process. In model II the first step represents the conformational change of the oligomer and the second step the dissociation of this species with the concomitant unfolding process. A canonical ensemble was employed to describe these systems, considering that the total protein concentration remains constant. In the present work we show and analyze the behaviour of these systems in different conditions, and how could this analysis help with the identification of the unfolding mechanism experimentally observed. |
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BibTeX:
@Poster{Burgos128,
author = {Inés Burgos},
title = {A novel theoretical tool for the analysis of calorimetric data of oligomeric proteins},
booktitle = {Tuesday},
year = {128}
}
|
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| Thompson Clauyear Elizabeth | The alcohol dehydrogenase gene family: structural studies and the functional diversification process. | 129 | Monday | Poster | |
| Abstract: The alcohol dehydrogenase (ADH) proteins belong to the medium-chain dehydrogenases/reductases (MDR) superfamily which has almost 1000 members spread in all types of organisms, and they have been implicated, additionally to the ethanol oxidation, in norepinephrine, dopamine, serotonin and bile acid metabolism, and oxidation of retinol in vitro and in vivo. Despite the fact this enzyme shows variable metabolic function, its three-dimensional structure is highly conserved. Different patterns of expression and differences in nonsynonymous substitution rates between gene copies are found in this family. The DNA and protein sequences were obtained from National Center of Biotechnology Information (NCBI). The ClustalW program was used to perform the alignments, which were inspected and manual changes made when necessary, using GeneDoc (Multiple Sequence Alignment Editor & Shading Utility). The estimation of the phylogenies were conducted by neighbor-joining (NJ), using the p distance and the Poisson-corrected amino acid distances to analyze the amino acid sequences, and the Kimura twoÂparameter method as substitution model to the DNA sequences, available in the MEGA program version 3.1 (Molecular Evolutionary Genetics Analysis), and by maximum likelihood (ML) methods using PhyML and TreeFinder programs. In total, 1500 repetitions were performed using the bootstrapping method to determine the reliability of each node of the tree. The coefficients of functional divergence (θ) of pairwise comparisons of the alcohol dehydrogenase gene family were estimated by the DIVERGE program. The program MODELLER was used for the construction of theoretical models of the 3D structure of the ADH proteins. PROCHECK and VERIFY-3D were used to validate the models. The phylogenetic analysis indicate that there have been several duplication events within plants, chordates, fungi, and bacteria, leading to the conclusion that the loci may not be homologous. No indication of positive selection was encountered for the Adh gene family. However, the coefficients of functional diversification estimated between the loci indicate statistically significant site-specific shift among them, as well as between those of different taxonomic families, suggesting that altered functional constraints may take place at some amino acid residues after speciation. The amino acid residues identified to be important for the functional diversification process were mapped onto the ADH three-dimensional structures. | |||||
BibTeX:
@Poster{Elizabeth129,
author = {Thompson Clauyear Elizabeth},
title = { The alcohol dehydrogenase gene family: structural studies and the functional diversification process.},
booktitle = {Monday},
year = {129}
}
|
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| Torres Jesus M | In silico analysis of different herpesviruses glycoprotein H (gH) | 131 | Monday | Poster | |
| Abstract: The members of Herpesviridae family require to fuse their viral envelope with the host cell membrane in order to begin the infection process. This fusion event is poorly understood and it depends on the presence and activity of three essential viral glycoproteins, gB and the complex formed by gH-gL. To date, only gB crystal structure has been determined. Due to the fact that gH-gL is needed to complete membrane fusion, bioinformatic methods were applied to determine the structural features in gH that may be related to its ability to act as a membrane fusion effector. An analysis of four gH from different herpesvirus infecting humans (HSV-1, VZV, HCMV and EBV) was carried out. A domain with similar secondary structure was found near the transmembrane domain in all the gH studied. In this domain, a possible fusion peptide and a region that may also be involved in membrane fusion due to its capacity to partition into membranes were found. Different hydrophobicity plots were determined for gH searching for hydrophilic regions which may correspond to possible antigenic sites. Several different hydrophilic regions were found for the gH of each virus studied, but one in particular, near the amino terminus of gH was found in all the glycoproteins, suggesting a common antigenic site. Only one 3D model of gH could be obtained by ab inito modelling (HSV-1) this model is the only one that correlates the secondary structure previously determined with its 3D structures. | |||||
BibTeX:
@Poster{M131,
author = {Torres Jesus M},
title = { In silico analysis of different herpesviruses glycoprotein H (gH)},
booktitle = {Monday},
year = {131}
}
|
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| César Díaz Pérez | MOLECULAR MODELLING OF MCC-CARBOXYLASE FROM Pseudomonas aeruginosa |
132 | Tuesday | Poster | |
| Abstract: A key enzyme in the assimilation of leucine in P. aeruginosa is the 3-methylcrotonyl- CoA carboxylase (MCCase), a biotin carboxylase (formed by ? and ? subunits), this enzyme uses ATP for the carboxylation of 3-methylcrotonyl-CoA (1). In this work we perform the homology modelling of both MCCase subunits, using the MODELLER software (2). The subunit ? was modelled dividing it in its principals domains: the biotin carboxylase domain (BC) and the biotin carboxylase carrier protein domain(BCCP). The BC domain was modeled using the Acetyl-CoA carboxylase from Escherichia coli (50% identity); our model presented the classic fold of the ATP-grasp superfamily. We found possible interactions between ATP and lysines 122 and 164; histidine 264, and glutamic acid 206. The modelling of BCCP domain was performed using the biotinyl domain of acetyl-CoA carboxylase from E. coli (35% identity); this structure was resolved whit the biotin, it allowed us to fit biotin in the biotin binding motif (AMKAM) present in the model. ? subunit was modeled using the transcarboxylase 12s from Propionibacterium freudenreichii (36% identity). We fit methylmalonyl-CoA to localize the active site in our model, it allowed us to identify the possible interacting residues with the coenzyme A. This is the first model of a MCCase; it will help us to understand the kinetics properties of this enzyme. 1. J. A. Aguilar et al., Appl. Environ. Microbiol. 72, 2070 (March 1, 2006, 2006). 2. A. Fiser, A. Sali, Methods in Enzymology 374, 461 (2003). |
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BibTeX:
@Poster{Perez132,
author = {César Díaz Pérez},
title = {MOLECULAR MODELLING OF MCC-CARBOXYLASE FROM Pseudomonas
aeruginosa},
booktitle = {Tuesday},
year = {132}
}
|
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| Torres-Gavilan Alejandro | Efective enzymatic resolution of phenylethylamine with Candida antarctica lipase B | 133 | Monday | Poster | |
| Abstract: An Âeasy-on, easy-off process for the effective resolution of (+/-)-1-phenylethylamine was completed using the lipase B of Candida antarctica. To our knowledge this is the first time a lipase is used in a two step method for the resolution of a chiral arylalkilamine involving a high-conversion enantioselective condensation of (R)-(+)-1-phenylethylamine with capric acid (99% yield, <24h), followed by the hydrolysis of the corresponding synthesized (R)-(+)-amide (97% yield, 48h). As a result, this efficient process yielded both (R) and (S) enantiomers of 1-phenylethylamine in high enantiomeric purity. | |||||
BibTeX:
@Poster{Alejandro133,
author = {Torres-Gavilan Alejandro},
title = { Efective enzymatic resolution of phenylethylamine with Candida antarctica lipase B},
booktitle = {Monday},
year = {133}
}
|
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| Trejo-Garcia M | Docking in triosephosphate isomerase from Leishmania mexicana. | 135 | Monday | Poster | |
| Abstract: Leishmania is a parasitic protozoa representative of the family Trypanosomatide which causes a disease widespread in many tropical and subtropical regions of the world where they constitute a serious health problem. According to the World Health Organization, 12 million people in the world suffer leishmaniasis. The drugs for the treatment exhibit serious side effects such as renal and caryearc toxicity. Therefore, there is an urgent need for the development of new, efficient, and safe drugs for the treatment of this disease. One approach in the search of new drugs is to found molecules that inhibit an enzyme from parasite. Glycolysis plays an essential role in ATP supply in trypanosomatides. Triosephosphate isomerase (TIM) is a homodimeric glycolytic enzyme that catalyzes the interconversion between glyceraldehyde-3-phosphate and dihydroxyacetone phosphate and is active only in its dimeric form. Therefore, the binding at the dimer interface of some organic molecules could affect the interactions between the two monomers of TIM, and cause its inhibition. In this context, using a molecular simulation approach (docking), we studied the binding of around 50,000 compounds at the dimer interface of TIM from L. mexicana (LmTIM). The data showed that the residues Tyr102, Glu104, Lys112 from one monomer and Tyr101, Tyr 102, Arg98, Glu104, Glu77, Lys112 from the other monomer were important for the binding of these molecules at the LmTIM interface. Also, in vitro assays were made to determine the effect on LmTIM activity of the one hundred compounds with the highest binding energy according to the docking. | |||||
BibTeX:
@Poster{M135,
author = {Trejo-Garcia M},
title = { Docking in triosephosphate isomerase from Leishmania mexicana.},
booktitle = {Monday},
year = {135}
}
|
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| Trivella Daniela Barretto Barbosa | Structural aspects of genistein binding to human transthyretin (TTR) | 137 | Monday | Poster | |
| Abstract: Transthyretin (TTR) is a tetrameric β-sheet rich protein, involved in the transport of thyroid hormone thyroxine (T4) and retinol. The tetrameric assembly of TTR can dissociate into monomers and these spontaneously self-assemble to form amlyloid fibrils. The later fibrils and monomeric intermeyeartes of TTR are related to a group of diseases collectively known as Sistemic Amyloidosis (SA). Small molecules, both of synthetic and natural origin, are able to fit into the two identical T4 binding sites strongly delaying TTR tetramers dissociation and fibril formation. This fact prompted active the search of pharmacophores that can be useful for TTR amyloidosis treatment. Recently it was shown that soybean isoflavone genistein displays a potent inhibition of TTR fibril formation, and is therefore an interesting candidate for SA treatment. Genistein and other TTR ligands bind to the two T4 binding sites in a negative cooperative process through an unclear mechanism. Here we describe the crystallographic structures of wild type and V30M TTR mutant bound to genistein. Detailed analysis of the crystal structure of wild type TTR revealed new insights into the molecular aspects of negative coopertativity displayed by TTR ligands. Ser117 side chain and structurally positioned water molecules plays an important role in ligand binding and allosterism. However, the V30M mutation affects negative allosteric changes caused by genistein binding to TTR and influences a conformation in the FG loop. Collectively, these results clarify the molecular basis of allosterism exhibited by geinstein when bound to TTR. Financial support: FAPESP CNPQ |
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BibTeX:
@Poster{Barbosa137,
author = {Trivella Daniela Barretto Barbosa},
title = { Structural aspects of genistein binding to human transthyretin (TTR) },
booktitle = {Monday},
year = {137}
}
|
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| Gabriel Gutiérrez Magdaleno | Redesign of the Lysozyme's recognition site | 138 | Tuesday | Poster | |
| Abstract: In recent years, our research group has been studying protein-carbohydrate (P-C) interactions, trying to clarify the underlying energetic-structural principles of the phenomenon. As a new stage in our study of P-C complexes, we have aimed to redesign the proteins recognition site, so that its specificity is changed towards other saccharidic structures. The primary target of the present project is to redesign the recognition site of hen ?s egg lysozyme to make it specific for glucose ?(1-4) oligomers. Using the crystal structure of lysozyme with chitotriose (GlcNAc?(1-4)GlcNAc?(1-4)GlcNAc) as a template, a model of lysozyme binding cellotriose (Glc?(1-4)Glc?(1-4)Glc) was built, simply by eliminating the NAc moiety. After several cycles of mutations and energy minimizations Using Rosetta, a construct was obtained whose binding energy was comparable to that of the wild lysozyme-chitotriose complex. Mutations that optimized interactions with the OH2 groups in cellotriose were: Ile98Gln, Ile58Gln, Leu56Ser, Trp108Tyr, Leu75Arg. Furthermore, the Ala107Asn, Trp63Tyr mutations achieved increased affinity with common groups in cellotriose and chitotriose. Subsequently, the expression of recombinant lysozyme in Aspergillus niger was implemented in our lab. The recombinant protein was characterized by means of circular dichroism and fluorescence spectroscopy. In addition, the binding with chitotriose was measured calorimetrically. Overall, the secondary and tertiary structures, the thermal stability and the binding properties of recombinant lysozyme were identical to those of lysozyme purchased from SIGMA. |
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BibTeX:
@Poster{Magdaleno138,
author = {Gabriel Gutiérrez Magdaleno},
title = {Redesign of the Lysozyme's recognition site},
booktitle = {Tuesday},
year = {138}
}
|
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| Uriostegui-Alejo LA | Protein-protein interaction of human eIF3f | 139 | Monday | Poster | |
| Abstract: INTRODUCTION. The eukaryotic initiation factor 3f (eIF3f) is the p47 subunit of the multi-subunit eIF3 complex, which plays an important role in translation initiation. The apparent molecular weight of human eIF3f is 47 kDa (1). Results from our lab showed that eIF3f is a cell cycle related protein, the silencing of its expression opens death programs and we know that it is a negative regulator of protein synthesis in vitro as well as in vivo (2,3). Our interest is to know if eIF3f forms part of other multi-subunit complexes. METHODS. Total protein extracts were obtained from A459 cells and immunodetection of eIF3f was assayed on native PAGE (Polyacrylamide gel electrophoresis) to observe different multi-subunit complexes. One of the multi-subunit complexes was isolated and fixed by different methods, such as cross-linking. The multi-subunit complex was separated in a specific manner and the proteins involved were determined. RESULTS. The native PAGE showed a pattern of different multi-subunit complexes present in the total protein extract. The immunodetection showed the presence of complexes that fluctuate from 100 to 1000 kDa. One of these was selected and analyzed uncovering interesting protein interactions. CONCLUSIONS. The eIF3f protein was identified interacting with high and low molecular weight multi-subunit complexes. REFERENCES 1. Asano K et al., 1997. J Biol Chem. 272: 27042. 2. Higareda AE et al., 2003. Nature Biotech. Short Reports. 14: 111. 2. Shi J et al., 2006. Oncogene. 25: 4923. | |||||
BibTeX:
@Poster{LA139,
author = {Uriostegui-Alejo LA},
title = { Protein-protein interaction of human eIF3f},
booktitle = {Monday},
year = {139}
}
|
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| Yobana Perez | Purificación de la Proteína recombinante rPv200L de Plasmodium vivax | 140 | Tuesday | Poster | |
| Abstract: La proteína rPv200L es un fragmento recombinante de la región N-terminal de la proteína de superficie de merozoito 1 (MSP?1) de Plasmodium vivax, con características inmunológicas importantes, que la convierten en un blanco de estudio prometedor para el desarrollo de una vacuna antimalárica. En este trabajo se desarrolló una metodología de purificación basada en tres pasos cromatográfìcos con la que se puede obtener una proteína homogénea, cuya pureza es superior al 95%. Con esta proteína se podrán realizar pruebas bioquímicas e inmunológicas de alta confiabilidad; simultáneamente se implementaron técnicas de monitoreo sensibles a la detección de contaminantes provenientes del hospedero. Anticuerpos policlonales provenientes de suero de monos infectados con P. vivax, reconocen la proteína recombinante obtenida bajo estas condiciones. Complementario a este trabajo se analizaron las propiedades fisicoquímicas y se hizo la predicción de la estructura secundaria y terciaria de la proteína, mediante herramientas bioinformáticas. |
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BibTeX:
@Poster{Perez140,
author = {Yobana Perez},
title = {Purificación de la Proteína recombinante rPv200L de Plasmodium vivax},
booktitle = {Tuesday},
year = {140}
}
|
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| Utrilla-Coello R. G. | STARCH BIOSYNTHESIS ENZYMES OF PIGMENTED MAIZE: ELECTROPHORETIC STUDY | 141 | Monday | Poster | |
| Abstract: In recent years, our understanding of starch structure and our knowledge of the enzymes that are involved in starch biosynthesis has increased greatly because these define their molecular structure. Although in the starch biosynthesis approximately thirteen enzymes are involved, only three enzymes are considered key points in the starch synthesis: starch synthase (SS), starch branching enzyme (SBE) and starch debranching enzyme (SDBE). In this sense, the aim of this work was carried out an electrophoretic study of the starch biosynthesis enzymes of blue and white maizes. Starch was isolated from both varieties and starch biosynthesis enzymes were extracted using diverse buffers and tensoactive agents. SDS-PAGE and 2D-PAGE were carried out. The SDS-PAGE pattern of both maize starches was different. The SDS-PAGE of blue maize starch showed 60, 90 and 120 kDa proteins that correspond to biosynthesis enzymes GBSSI, SBE and SSSII, respectively. The white maize starch did not show 120 kDa protein. The expression of GBSSI in blue maize starch is higher than white maize starch. The 2D-PAGE only showed approximately 7 spots of 60 kDa protein (GBSSI) with pI range between 5 and 6; this is the predominant enzyme associated to starch granule. The differences in the electrophoretic pattern suggest that starch biosynthesis in both maize starches differ, therefore structural differences might be present and consequently they will show different functional properties. | |||||
BibTeX:
@Poster{G.141,
author = {Utrilla-Coello R. G.},
title = { STARCH BIOSYNTHESIS ENZYMES OF PIGMENTED MAIZE: ELECTROPHORETIC STUDY},
booktitle = {Monday},
year = {141}
}
|
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| Valadares Napoleao F | Surface Plasmon Resonance studies of thyroid hormone receptor beta | 143 | Monday | Poster | |
| Abstract: The thyroid hormones influence the lipid metabolism and the cardiovascular function. Most of the actions of the thyroid hormones are meyearted by the thyroid hormone receptors, TRα and TRβ. Some effects of thyroid hormones might be therapeutically useful in several disorders if adverse effects can be minimized or eliminated. Compounds that discriminate between these subtypes may be useful for the treatment of important medical problems such as obesity and hypercholesterolemia. However, the classic methodology to detect the interaction between the thyroid hormone receptors (TRs) and its ligands depends upon expensive techniques, like the classic radio-ligand assay, which detects direct interactions between ligands and receptors, or the reporter gene transfection assay. The thyroid hormone receptors undergo a conformational change upon agonist ligand binding, and only after this change the surface for recognition and binding of coactivators that regulate hormone dependent transcriptional activity is formed. Thus, itÂs possible to determinate if a ligand has agonist activity through the detection of the interaction between the complex TR-agonist with its coactivator. In the present study we sought to detect these receptor-coactivator interactions induced by agonist ligands using surface plasmon resonance, a methodology that allows the detection of TR-agonist and coactivator interactions in real time, requires low sample amounts, avoid the need of radio-labeled ligands and allow the detection of agonist activity of new TR ligands. |
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BibTeX:
@Poster{F143,
author = {Valadares Napoleao F},
title = { Surface Plasmon Resonance studies of thyroid hormone receptor beta},
booktitle = {Monday},
year = {143}
}
|
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| Torres Flores Jesús M | BIOINFORMATICAL ANALYSIS OF GLYCOPROTEIN H (GH) BELONGING TO DIFFERENT MEMBERS OF THE Herpesviridae FAMILY | 144 | Tuesday | Poster | |
| Abstract: Members of the Herpesviridae Family need to fuse the viral envelope with the host´s cell membrane in order to begin the infection process. This fusion event is poorly understood and it depends on the presence and activity of 3 essential viral glycoproteins gB and the complex formed by gH-gL. To date, only gB crystal structure has been determined. Due to the fact that gH-gL is needed to complete membrane fusion, we applied bioinformatic methods to determine diferrent structural features in gH that may confere it the abbility to be the membrane fusion effector. An analysis of four gH belonging to different herpes virus infecting humans (HSV-1, VZV, HCMV and EBV) was carried out. A domain with similar secondary structure was found near the transmembrane domain in all the gH studied. In this domain, we found a possible fusion peptide and a region that may also be involved in membrane fusion due to its capacity to partition into membranes. Different hydrophobicity plots were determined for gH to search for hydrophilic peaks on the plot which may correspond to possible antigenic sites. Several different hydrophilic regions were found for the gH of each virus studied, and one in particular, near the amino terminus of gH was found in all the glycoproteins analyzed possibly indicating a common antigenic site. Only one 3D model of gH could be obtained by ab inito modelling (HSV-1) this model is the only one that correlates the secondary structure previously determined with its 3D coordinates | |||||
BibTeX:
@Poster{M144,
author = {Torres Flores Jesús M},
title = {BIOINFORMATICAL ANALYSIS OF GLYCOPROTEIN H (GH) BELONGING TO DIFFERENT MEMBERS OF THE Herpesviridae FAMILY},
booktitle = {Tuesday},
year = {144}
}
|
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| Valdez Miguel A. | Interfacial behavior of N- Nitrosodiethylamine- Serum Bovine Albumin complexes with DPPC at the air -water and water-chloroform interfaces | 145 | Monday | Poster | |
| Abstract: We report the interfacial interaction of albumin (BSA) and the phospholipid (DPPC) at the air Âwater interface by the Wilhelmy plate technique with a Langmuir Blodgett balance. Dilatational Modulus and interfacial tension measurements of the BSA Â N - Nitrosodyethilamine at the air-water and chloroform-water interface were performed with a drop tensiometer. Measurements of the BSA-Nitrosodiethylamine adsorption at the DPPC monolayer were performed for different carcinogen concentrations. We analyze the adsorption of BSA from the aqueous subphase on a DPPC monolayer lying at the air water interface at different pressures of the monolayer. Differences of the BSA adsorption depended on the concentration of the carcinogen and isotherms modifications were observed. Brewster angle microscopy images were used to follow the BSA- carcinogen adsorption. The protein conformation was investigated in solution and in a DPPC-BSA solid film by circular dichroism. Fluorescence for the carcinogen-albumin solutions were also determined. The results show that carcinogens strongly interacts with the albumin structure and the adsorption at the interface is modified because of the carcinogen molecules attached to the BSA molecules. However, the secondary structure in solution is almost not modified. We obtain the free energy of the carcinogen ÂBSA interaction indirectly by using fluorescence spectroscopy . Measurements of dynamic interfacial tension, dilatational storage and loss modulus were also performed by using a drop tensiometer at different frequencies. Surface tension and the elastic modulus were obtained for different carcinogen concentrations. | |||||
BibTeX:
@Poster{A.145,
author = {Valdez Miguel A.},
title = { Interfacial behavior of N- Nitrosodiethylamine- Serum Bovine Albumin complexes with DPPC at the air -water and water-chloroform interfaces},
booktitle = {Monday},
year = {145}
}
|
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| F Zárate-Pérez | Characterization of the equilibrium unfolding/refolding reaction of the monomeric Triosephosphate isomerase from T. cruzi |
146 | Tuesday | Poster | |
| Abstract: Stability and thermodynamic parameters of triosephosphate isomerase (TIM) have been analyzed widely. However, in many cases these studies show difficulties to understand accurately intrinsic or subtle changes during the folding/unfolding process. The dimeric triosephosphate isomerase of T. cruzi, (TcTIM) presents two intermediates in its folding/unfolding pattern, one of them monomeric. We have generated by genetic engineering a monomeric version of TcTIM (monoTcTIM), in order to understand its role in the stability of the homodimer. Folding pattern of this monoTcTIM was followed by spectroscopic techniques (circular dichroism and fluorescence). All the experiments were performed at equilibrium, using Gdn? HCl as denaturant. MonoTcTIM showed a reversible folding process. Experiments of intrinsic fluorescence showed a monophasic unfolding/refolding pattern. In the range of 0?6M of denaturant concentration intrinsic fluorescence intensity was increased by ANS binding. Results confirm the presence of the monomeric form of this mutant. The unfolding/refolding pattern and the equilibrium constant were also obtained. |
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BibTeX:
@Poster{Zarate?Perez146,
author = {Zárate?Pérez, F},
title = {Characterization of the equilibrium unfolding/refolding reaction of the monomeric
Triosephosphate isomerase from T. cruzi},
booktitle = {Tuesday},
year = {146}
}
|
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| Verdel-Aranda K | CHARACTERIZATION OF PHOSPHORIBOSYL ANTHRANILATE ISOMERASES (PRAI) OBTAINED BY NOVEL DIRECTED EVOLUTION APPROACHES | 147 | Monday | Poster | |
| Abstract: The (??)8-enzyme phosphoribosyl anthranilate isomerase (PRAI) catalyses the third step of tryptophan biosynthesis in most organisms. PRAI, encoded by the trpC gene in Escherichia coli, catalyses a general acid-base reaction. The functional versatility of the (??)8-scaffold as a whole, and the simplicity of the reaction catalyzed by PRAI, makes this protein scaffold a suitable model to explore novel directed evolution approaches. Indeed, we have isolated numerous PRAI variants that were generated using novel approaches, by means of complementation of a trpC minus E. coli auxotrophic strain. Although an enzyme assay to measure PRAI in vitro has been reported, such assay is not trivial due to the instability of the substrate PRA. A coupled assay involving enzymes that are not commercially available is the sole alternative. In this system, the enzyme anthranilate phosphoribosyl transferase (AnPRT) synthesizes the substrate PRA from anthranilate and PRPP. We have cloned and overexpressed the thermostable AnPRT from Sulfolobus solfataricus and will report in the optimization of the expression conditions. The downstream enzyme required to allow steady-state enzyme kinetics is indole glycerol phosphate synthase (IGPS), which uses the product of PRAI, CdRP, as substrate. IGPS from E. coli has also been cloned and purified. Using this enzyme assay involving AnPRT and IGPS, we are analyzing the newly evolved activities of selected PRAIs. We anticipate that a detailed kinetic characterization of these variants will allow us to interpret the evolutionary pathways followed in our directed evolution experiments, such that the approaches adopted can be understood and iteratively improved. | |||||
BibTeX:
@Poster{K147,
author = {Verdel-Aranda K},
title = {CHARACTERIZATION OF PHOSPHORIBOSYL ANTHRANILATE ISOMERASES (PRAI) OBTAINED BY NOVEL DIRECTED EVOLUTION APPROACHES},
booktitle = {Monday},
year = {147}
}
|
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| Vigueras-Ceballos Alan | Protein-ligand interaction of periplasmic binding protein LAO, and the roll of the hinge region in the affinity for its substrates | 149 | Monday | Poster | |
| Abstract: The periplasmic binding proteins (PBP) are soluble proteins which participate in molecular recognition of diverse ligands in process like transport and chemotaxis (Ames and Lever, 1972). PBP`s belong to a super family that shows grate diversity in sequence but conserves their tertiary structure (Dwyer y Hellinga, 2004),the global topology of PBP`s contain two α/β domains joint together by a hinge region, the ligand-binding site lies in the interface of the two domains.. (Nikaido and Ames, 1992.) LAO protein has grate affinity for the amino acids Lysine15 nM, Arginine 14 nM and Ornithine 30 nM (Chul-Hee Kang et all, 1991). It display two states, in presence of ligands is closed, and in the absence of ligands is open (Oh et all, 1994). Maintaining intact the protein-ligands direct interactions, the affinity of LAO can be manipulated by altering the intrinsic equilibrium between the open and close state, making punctual mutations in hinge region and introducing functional groups whit different characteristics, modify residues that make contacts between domains, representing changes in the affinity for the substrate and diminish the affinity constant for each ligand. ÂAmes, G. F.-L., and Lever, J. E. (1972) J. Biol. Chem. 247,4309-4316. Â Dwyer M. A., Hellinga H. M.; (2004), Current opinion in structural biology, 14:495-50. ÂKang, C.-H., Shin, W.-C., Yamagata, Y., Gokcen, S., and Ames, G. F.-L., and Kim, S.-H. (1991) J. Bwl. Chem. 266,23893-23899. ÂNikaido, K., and Ames, G. F.-L. (1593) J. Biol. Chem. 267,2070~20712. ÂOh, B.-H., Pandit, J., Kang, C.-H., Nikaido, K., Ames, G. F.-L., and Kim, S.-H. (1993) J. Biol. Chem 268,11348-11355. |
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BibTeX:
@Poster{Alan149,
author = {Vigueras-Ceballos Alan},
title = { Protein-ligand interaction of periplasmic binding protein LAO, and the roll of the hinge region in the affinity for its substrates},
booktitle = {Monday},
year = {149}
}
|
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| Ana Maria Anaya Tapia | Denaturation/renaturation of triosephosphate isomerase in urea. Equilibrium studies | 150 | Tuesday | Poster | |
| Abstract: In this work we study the reactions of unfolding and refolding of TIM of T. cruzi (TcTIM) induced by urea in an interval of 0 to 9.0 M, at 25 ° C. The change in Intrinsic Fluorescence as a function of time shows that a suitable time to reach equilibrium is 200 hours for the denaturalization and 100 hours for the renaturalization. Samples of increasing denaturant concentration were equilibrated and analyzed by changes in fluorescence intensity, and catalytic activity. The results indicate that the denaturation pattern of TcTIM in urea is not a simple two state process, and that at least two step appears in the reaction i.e. presents a clear intermediate region. This conformer binds ANS in an interval of urea of 0.4-0.8 M. On the other hand, the reversibility of the process was investigated; recovery of catalytic activity shows similar values as observed in the native enzyme. This observation is interesting because triosephospate isomerase from T. brucei is very similar in sequence (74 % identity) and three dimensional structure (rms < 1 A) to TcTIM. These differences are excellent to study folding relationships between oligomeric homologous proteins. | |||||
BibTeX:
@Poster{Tapia150,
author = {Ana Maria Anaya Tapia},
title = {Denaturation/renaturation of triosephosphate isomerase in urea. Equilibrium studies},
booktitle = {Tuesday},
year = {150}
}
|
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| Zamudio Ocadiz Jose N | IMMOBILIZATION OF TEM-1 B-LACTAMASE INTO SOL GEL MATRIX FOR BIOSENSOR DEVELOPMENT | 151 | Monday | Poster | |
| Abstract: We have immobilized the wild type TEM-1 lactamase and mutants labeled with fluorophores into sol-gel matrices for detection of penicillin in solution. The incorporation of proteins into sol-gel matrices have been used for biosensor development since proteins retains their activity and are more stable to the thermic unfolding. The advantage of this technique is that the matrix is invisible to different spectroscopic techniques such as uv-visible spectroscopy, fluorescence, circular dicrhoism, paramagnetic techniques and infra red spectroscopy that allow the study of proteins-ligand interaction into sol-gel materials which is the basis of biosensor development. In general the detection of small ligand by proteins have been exploited the conformational changes upon ligand binding, however TEM-1 -lactamase does not carry any conformational change after ligand interaction. In this work we have been labeling with fluorophores the mutant Glu166Cys of the active site, since It have been reported that the incorporation of labels at the specific position of the active site of this enzyme (1,2), is possible to detected the antibiotic interaction with TEM-1 -lactamase. The advantage of use Glu166Cys mutant is that the catalysis is impaired, the deacylation step becomes slow, therefore the majority of the enzyme is in the acylated form, upon ligand binding, which combined with the specific incorporation of fluorophores at this position is possible to calibrate the penicillin in solution by the changes in the fluorescence. 1. Cha, P-H., et al. (2004). J. Am. Chem. Soc. 126, 4074-4075. 2. Mustafi, D, et al. (2001). Biochemitry, 40, 2397/2409. |
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BibTeX:
@Poster{N151,
author = {Zamudio Ocadiz Jose N},
title = { IMMOBILIZATION OF TEM-1 B-LACTAMASE INTO SOL GEL MATRIX FOR BIOSENSOR DEVELOPMENT},
booktitle = {Monday},
year = {151}
}
|
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| Francisco Sandoval Becerril | Effect of the ionic strenght in the folding/unfolding reactions of the triosephosphate isomerase from Trypanosoma cruzi | 152 | Tuesday | Poster | |
| Abstract: The triosephospahte isomerase (TIM) is an enzyme that catalyzes the fifth step of glycolysis and is a model very utilized in the folding studies for his ubiquity in the nature. The TIM from Trypanosoma cruzi is a dimeric protein and has a structure called barrel (?/?)8. TcTIM has a folding process of four states in guanidinium hydrochloride (Chánez-Cárdenas, 2005) described by: N2 ? N2 * ? 2M ? 2U Were: N2 Is the native dimeric native state N2 * Is the non-native dimeric native state 2M Is the monomeric state 2U Is the unfolding state In equilibrium experiments in low guanidinium hydrochloride concentrations (0.2-1 M); it has been observed that there is an increase in the catalytic activity with respect to the native folding state. In this work we study the effect of ionic strength in the folding/unfolding reaction of TcTIM. The final intention of this investigation is to know if the ionic strength has an direct effect of the increase in the catalytic activity observed under guanidinium chloride incubation. References: Chánez-Cárdenas ME, Pérez-Hernández G, Sánchez-Rebollar BG, Costas M, Vázquez-Contreras E. 2005. Reversible equilibrium unfolding of Triosephosphate isomerase from Trypanosoma cruzi in guanidinium hydrochloride involves stable dimeric and monomeric intermediates. Biochemistry 44:10883-1092. |
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BibTeX:
@Poster{Becerril152,
author = {Francisco Sandoval Becerril},
title = {Effect of the ionic strenght in the folding/unfolding reactions of the triosephosphate isomerase from Trypanosoma cruzi},
booktitle = {Tuesday},
year = {152}
}
|
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| Arabolaza A | Determinants of substrate specificity in the ? subunit of Acyl-CoA Carboxylases from S. coelicolor | 153 | Monday | Poster | |
| Abstract: Acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC) of Streptomyces coelicolor are multisubunit complexes, constituted by three different subunits ?, ? y ?. ACC and PCC catalyze the carboxylation of acetyl- and propionyl-CoA to generate malonyl- and methylmalonyl-CoA, respectively. These molecules are essential building units for straight and branched fatty acids and polyketide biosynthesis. The core catalytic ? ??subunits, PccB and AccB (for ACC and PCC respectively), catalyze the transcarboxylation between carboxibiotin and acyl-CoAs. The crystal structure of PccB showed a hydrophobic and highly conserved biotin-binding site and defined for the first time the propionyl-CoA binding pocket. Based on the sequence alignment and structure comparison between AccB and PccB we identify some active site key residues involved in the acyl-CoA binding. The influences of these aminoacids in determine substrate specificity was investigated by site directed mutagenesis, biochemical characterization of the mutant proteins and direct visualization of the active site by crystallization of the variants generated. Principally we performed saturation mutagenesis of Asp(422) of PccB, positioned at the ?end? of the acyl-CoA binding pocket. We determined the catalytic parameters and have already crystallized and solved the crystal structural data of six novel PccB mutants. Interestingly by structural comparison of the active site between PccB and each of the mutants we observed extensive conformational changes in residues located ?near the entrance? of the acyl-CoA binding pocket that clearly impinge on the substrate setting. Understanding the substrate specificity of these enzymes will help to facilitate bioengineering to provide novel extender units for polyketide biosynthesis. |
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BibTeX:
@Poster{A153,
author = {Arabolaza A},
title = {Determinants of substrate specificity in the ? subunit of Acyl-CoA Carboxylases from S. coelicolor},
booktitle = {Monday},
year = {153}
}
|
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| Martiniano Bello Ramírez | Energetics of bovine ?-lactoglobulin homodimerization | 155 | Monday | Poster | |
| Abstract: The interface of ?-lactoglobulin (?lg) homodimer portrays stereochemical properties (v.gr., small size, large polarity and high residual hydration) that appear rather non-standard in relation to most protein homodimeric interfaces characterized so far. To provide new insights into the energetic bases that govern the formation of this ?atypical? homodimer, the dissociation process of bovine ?lg variant A at a neutral pH was characterized here thermodynamically by conducting dilution experiments with an isothermal titration calorimeter. Association was enthalpically driven throughout the temperature range spanned. The homodimer?s maximum stability was observed at 25 °C. The heat capacity change was significantly more negative than estimates based on surface area changes, suggesting the occurrence of effects additional to the dehydration of the contact surfaces between subunits. Near-UV CD spectra proved to be independent of protein concentration, indicating that the protomers associated to each other following a rigid body-like behavior. Furthermore, the process proved not to be coupled to significant changes in the protonation state of ionizable groups or counterion exchange. In contrast, both osmotic stress experiments and a computational analysis of the dimer?s 3D structure indicated that a large number of water molecules are incorporated into the interface upon association. Numerical estimates considering the contributions of water immobilization and interface area desolvation accounted satisfactorily for the experimental heat capacity change. Thus, our study underlines the importance of explicitly considering the effects of water sequestering to perform a proper quantitative analysis of the formation of homodimers that preserve a large amount of residual hydration at the interface. |
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BibTeX:
@Poster{Ramirez155,
author = {Martiniano Bello Ramírez},
title = {Energetics of bovine ?-lactoglobulin homodimerization},
booktitle = {Monday},
year = {155}
}
|
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| Freitas M.S. | CHARACTERIZATION OF INTERACTION BETWEEN EBOLA FUSION PEPTIDE AND LIPID MEMBRANES | 157 | Monday | Poster | |
| Abstract: Ebola viruses are filamentous and enveloped that belongs to Filoviridae family. The lipid envelope contains one type of protein (Ebola GP) arranged into oligomers. The Ebola GP is responsible for both receptor binding and membrane fusion. It is proteolytically cleaved into disulfide-linked GP1 and GP2 subunits. The N-terminal domain of glycoprotein (GP2) is the fusion peptide region, which is thought to insert directly into the target membrane. The main goal of this work is to characterize the interaction between the fusion peptide and cellular membranes. The interaction between Ebola fusion peptide with VERO cells induced lipid mixing of cellular membranes. However, the Ebola peptide was not able to induce lipid mixing of cholesterol-depleted VERO cells indicating that cholesterol is playing a crucial role in membrane fusion. To understand the importance of cholesterol, we isolated membrane microdomains, named lipid rafts. First over all, we analyzed the peptide-lipid interaction by NMR. The chemical shift of 1H resonances suggest that the fusion peptide interact with lipid rafts. Besides, the peptide induced lipid rafts aggregation, indicating fusion among rafts. In order to understand the interaction between Ebola fusion peptide and membranes we used in silico approaches. The data show that Ebola fusion peptide interacts with membranes because of the hydrophobicity of the carbonic skeleton of lipids, although the structure is stabilized by polar head of lipids. After that, we showed the interaction between aromatic-aromatic residues that probably contributed to structure stability. This work is the first indicative of direct interaction between Ebola fusion peptide and lipid rafts, a crucial information since the fusion peptide is a potential target to the development of antiviral drugs. | |||||
BibTeX:
@Poster{M.S.157,
author = {Freitas M.S.},
title = {CHARACTERIZATION OF INTERACTION BETWEEN EBOLA FUSION PEPTIDE AND LIPID MEMBRANES},
booktitle = {Monday},
year = {157}
}
|
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| Mariana Pierre de Barros Gomes | Characterization of the PrP:RNA complex: structural insights and toxicity studies | 157 | Monday | Poster | |
| Abstract: Prion diseases are neurodegenerative and fatal diseases that affect mammals and can be inherited, sporadic or infectious. Their occurrence is related to a misfolded isoform of the normal cellular protein, (PrPC), the Scrapie prion protein (PrPSc). PrPC is normally attached to the plasmatic membrane of neurons with an ?-helical rich structure. PrPSc has a ?-sheet rich structure, aggregates and is resistant to treatment with proteases. The ?protein only hypothesis? postulates that the PrPSc is the only agent responsible for occurrence of prion diseases, acting as a template to PrPC conversion, generating more PrPSc, but there are evidences that an adjuvant factor may be involved, lowering the energy barrier between the two isoforms. We have identified that interaction of PrP with double-stranded DNA lead to a higher ?-sheet content, with similar characteristics of PrPSc and have structurally characterized the PrP:DNA complex. RNA molecules can also interact with PrP and potentially modulate PrPPC?PrPSc conversion. In this work we have investigated the interaction of recombinant full-length PrP and two PrP amino-terminal deletion mutants with distinct RNA sequences or total RNA extracted from cultured cells. We have shown that PrP interacts with RNA with nanomolar affinity and aggregates upon this interaction. RNA does not induce aggregation of the PrP N-terminal deletion mutants, indicating that the N-terminal region is important for this process. Cell viability assays showed that PrP:RNA aggregates can affect cultured cells. These results indicate that RNA can modulate recombinant prion protein conversion into toxic aggregates. |
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BibTeX:
@Poster{Barros157,
author = {Mariana Pierre de Barros Gomes},
title = {Characterization of the PrP:RNA complex: structural insights and toxicity studies},
booktitle = {Monday},
year = {157}
}
|
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| María Natalia Lisa | Metal Substitution in GOB-18 Metallo-?-lactamase | 159 | Monday | Poster | |
| Abstract: Metallo-?-lactamases (M?Ls) are zinc dependent enzymes able to hydrolyze and inactivate most ?-lactam antibiotics. The large diversity of active site structures and metal content among M?Ls from different sources has limited the design of a pan-M?L inhibitor. These enzymes have become of great concern in the medical area as they are becoming widely distributed among pathogens. GOB is a M?L produced by the opportunistic pathogen Elizabethkingia meningoseptica, causative agent of meningitis. Recently, our group has reported the biochemical and biophysical characterization of GOB-18 (1). The studies performed showed that this enzyme is a mono-Zn(II) broad spectrum lactamase. Being that Zn(II) is a silent ion for most spectroscopic techniques, we have used different metal ions as probes for the study of the active site of GOB-18. Here we present the characterization of different metal derivatives of this novel M?L. [1] Moran-Barrio, J.; González, J.M.; Lisa, M.N.; Costello, A.L.; Dal Peraro, M.; Carloni, P.; Bennett, B.;Tierney, D.L.; Limansky, A.S.; Viale, A.M.; Vila, A.J. (2007). J. Biol. Chem. 282, 18286-18293. |
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BibTeX:
@Poster{Lisa159,
author = {María Natalia Lisa},
title = {Metal Substitution in GOB-18 Metallo-?-lactamase},
booktitle = {Monday},
year = {159}
}
|
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| Juan S. Martinez | INHIBITING THE ANAPHASE-PROMOTING COMPLEX: CHARACTERIZATION AND REGULATION OF ACM1 |
161 | Monday | Poster | |
| Abstract: The anaphase-promoting complex (APC) is a master regulator of the cell cycle. It is an ubiquitin ligase that targets specific substrates to the 26S proteasome for proteolysis. Substrate specificity and timing of APC activity are cell-cycle dependent and defined by binding of the co-activators Cdc20 or Cdh1. Using mass spectrometry we identified a novel protein complex that binds Cdh1 stably and inhibits APC-Cdh1. The complex includes the 14-3-3 homologs Bmh1 and Bmh2, and the previously uncharacterized protein we named Acm1. Formation of the complex is dependent on Acm1. Acm1 mediates association of the 14-3-3 proteins with Cdh1, and also regulates Cdh1 localization in the cell. Acm1 is expressed in a cell cycle-dependent manner, appearing at the G1/S transition and disappearing rapidly in late mitosis. We observed Acm1 stability is controlled by its phosphorylation status, and it is heavily phosphorylated in vivo. We mapped several consensus CDK sites by mass spectrometry, and found that full Acm1 phosphorylation was dependent on CDK function. A mutant Acm1 lacking CDK phosphorylation sites was constitutively unstable. We also observed that de- phosphorylation and proteolysis of Acm1 in vivo were dependent on Cdc14 phosphatase activity and that Acm1 was recognized as a substrate of Cdc14 in vitro. In addition, Acm1 was inappropriately degraded when ectopic overexpression of Cdc14 in S phase was induced. Our findings suggest that Acm1 stability is regulated by the opposing activities of Cdc28 and Cdc14, and define a mechanism for restricting protein expression to the time- frame corresponding to high CDK activity. |
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BibTeX:
@Poster{Martinez161,
author = {Juan S. Martinez},
title = {INHIBITING THE ANAPHASE-PROMOTING COMPLEX: CHARACTERIZATION AND
REGULATION OF ACM1},
booktitle = {Monday},
year = {161}
}
|
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| Mariana Paolorossi | Interaction of ?2-Glycoprotein I with Negatively Charged Phospholipid Membranes |
165 | Monday | Poster | |
| Abstract: ?2-Glycoprotein I (?2-GPI) is a soluble glycoprotein present in plasma. It participates in several physiological and pathogenic events. It was proposed, for example, that ?2-GPI binds to the exposed anionic phospholipids of apoptotic cells producing auto-antigenic lipid-protein complexes. The binding to the membrane is mediated by both electrostatic and hydrophobic forces. Even when the electrostatic interaction is absolutely required, the sole presence of a negative charge does not determine the strength of binding and different affinities are observed for different anionic lipids. We addressed the study of membrane interactions of ?2-GPI to understand the balance and mutual influence of electrostatic and hydrophobic driving forces and whether these interactions are coupled to conformational changes of the protein in the interface. ?2-GPI was purified from human plasma by heparin affinity chromatography. The interaction of ?2-GPI with phospholipid membranes composed of palmitoyl oleoyl phosphatidylglycerol (POPG), palmitoyl oleoyl phosphatidylserine (POPS) and palmitoyl oleoyl phosphatidylcholine (POPC) was studied by differential scanning calorimetry (DSC), fluorescence spectroscopy and infrared spectroscopy (FT-IR). The addition of negatively charged phospholipid membranes (POPG/POPC or POPS/POPC) to a pure ?2-GPI solution with low ionic strength, induced an increase in turbidity of the solution immediately after liposomes addition. Pure ?2-GPI exhibited a reversible two-state transition with a Tm of 64 ºC. In the presence of large unilamellar vesicles (LUVs) composed of POPC, the thermal unfolding was similar to that observed for pure ?2-GPI. When compared with that of the pure protein solution, the thermal transition of the protein in the presence of POPG/POPC or POPS/POPC LUVs presented remarkable differences which are partially abolished in a medium with high ionic strength (0.1M NaCl) . The secondary structure of ?2-GPI was studied by FT-IR and fluorescence spectroscopy. The presence of POPG/POPC or POPS/POPC LUVs induced a similar decrease in the fluorescence emission maximum wavelength in a medium of low ionic strength, but the infrared spectrum shape of the protein did not change significantly in the presence of POPG/POPC multilamellar vesicles between 25ºC and 80ºC. |
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BibTeX:
@Poster{Paolorossi165,
author = {Mariana Paolorossi},
title = {Interaction of ?2-Glycoprotein I with Negatively Charged
Phospholipid Membranes},
booktitle = {Monday},
year = {165}
}
|
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| Clarisa E. Alvarez | Reversible oxidation of maize recombinant C4 and non-C4 NADP-malic enzyme cysteine residues: implication in activity regulation [BibTeX] |
169 | Tuesday | Poster | |
BibTeX:
@Poster{,
author = {Clarisa E. Alvarez},
title = {Reversible oxidation of maize recombinant C4 and non-C4 NADP-malic enzyme cysteine residues: implication in activity regulation},
booktitle = {Tuesday},
year = {169}
}
|
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| Cristina Russo | Association of TATA Binding Proteins to DNA with Molecular Mechanics/PoissonBoltzmann Approach. |
171 | Monday | Poster | |
| Ricardo Perez | Site-directed mutagenesis of the conserved aromatic residues in the 5 tandem modules starch binding domain of Lactobacillus amylovorus alpha-amylase | 167 | Lunes | Poster | |
| Abstract: Alpha-amylase from Lactobacillus amylovorus contains five C-terminal repeated starch binding domains (SBDs) (Giraud, et al., 1997). The modules are identical and classified on the basis of primary structure into family 26 carbohydrate-binding modules (CAZY). In previous work it has been demonstrated the tandem modules are involved in the raw starch binding capacity of the enzyme (Rodríguez-Sanoja, et al., 2000). Polysaccharide-binding studies revealed that each module is able to adsorb to raw starch (Santiago, et al., 2005), and also an increase in the adsorption capacity as function of the number of modules, suggesting that each unit of the SBD may act in an additive or synergic way to optimize binding to raw starch (Guillén, et al., 2007). Sequence comparison of one of the modules forming the L. amylovorus alpha-amylase SBD revealed that Thr-11, Thr-32, Tyr-16, Tyr-18 and Tyr-20 in this domain are highly conserved in family CBM26 (Santiago, et al., 2005). Structural studies of the maltohexaose-forming amylase SBD from Bacillus halodurans (C-125) showed that the base of the binding is formed by one tryptophan (W36) and two tyrosines (Y23 and Y25) (Boraston, et al., 2006), correspondent into the L. amylovorus alpha-amylase SBD to W32, Y18 and Y20. To understand structure-function relationship in this multiple SBD, site-directed mutagenesis was conducted in one module, to replace in singles mutants the conserved threonines and tyrosines with leucines. Expressed proteins have been purified in nickel-chelate chromatography and assayed for binding capacity in affinity gel electrophoresis. The data showed that W32L mutant protein lost the capability to adsorb starch, whereas W11L did not affect the adsorption. We found also that Y18L and Y20L mutant proteins exhibit weaker affinity for starch. The other mutation Y16L did not show any effect on binding. In conclusion, one tryptophan residue, Thr32 homologous to Thr36 of the maltohexaose-forming amylase SBD from B. halodurans (C-125) is especially important because its mutation prevents adsorption; however the conserved tyrosines are necessary for full starch binding function of this SBD. References: Boraston, A. B., M. Healey, J. Klassen, E. Ficko-Blean, A. Lammerts van Bueren, and V. Law. 2006. J. Biol. Chem. 281:587-598. Giraud E. and G. Cuny. 1997. Gene 198:149-157. Guillén, D., Santiago, M., Linares, L., Pérez, R., Morlon, J., Ruiz, B., Sánchez, S. and Rodríguez-Sanoja, R. 2007. Submit Rodríguez-Sanoja, R., J. Morlon, J. Jore, J. Pintado, N. Juge, and J. P. Guyot. 2000. Appl. Environ. Microbiol. 66:3350-3356. Santiago, M., L. Linares, S. Sanchez, and R. Rodríguez-Sanoja 2005. Biología, Bratislava 60:111-114. |
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BibTeX:
@Poster{,
author = {Ricardo Perez},
title = {Site-directed mutagenesis of the conserved aromatic residues in the 5 tandem modules starch binding domain of Lactobacillus amylovorus alpha-amylase},
booktitle = {Lunes},
year = {167}
}
|
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| Abstract: TATA box binding proteins (TBPs) from hyperthermophilic and halophilic organisms, (e.g. Pyrococcus woesei (Pw) function optimally at high temperatures and/or salt concentration. Such proteins bind to DNA with different mechanisms than their mesophilic counterparts. Upon complex formation, the halophilic TBPs sequester cations while mesophilic TBPs release may both release and sequester ions from the DNA ion atmosphere. In this work, we have performed a computational study in order to understand the role of electrostatics on the binding of mesophilic and halophilic TBPs to DNA. |
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BibTeX:
@Poster{Russo171,
author = {Cristina Russo},
title = {Association of TATA Binding Proteins to DNA with Molecular Mechanics/PoissonBoltzmann
Approach.},
booktitle = {Monday},
year = {171}
}
|
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| Rodriguez Guadalupe C. | Expression of a recombinant antigen (Ov16) to Onchocerca volvulus | Tuesday | Poster | ||
| Abstract: The elimination onchocerciasis program in Mexico is based entirely on the use of ivermectin a drug that kills the skin microfilariae but not the adult worms. Thus, is important to carry out prospective entomological studies to evaluate the effect of ivermectin on transmission levels, and performing the monitoring for new infections in children, parasitologically or serologically, as an indicator of new infections. Although several antibody detection tests for onchocerciasis have been developed, the programs require accurate, sensitive, non-invasive, and 100% specific tests for onchocerciasis. A serologic test based on the use of a recombinant antigen (Ov16) in an ELISA protocol (using a conjugate to test for IgG4) is currently used by the onchocerciasis programs in Mexico and Guatemala. Given that the test requires Ov16 the objective of this study was to produce the recombinant protein using the binding protein maltose system. The production of the recombinant proteins was optimized, the resultant proteins were yearlysed to remove any traces of contaminating maltose, and the final concentration was determined using the Bradford technique. The optimum time to obtain the highest yield was three hours after induction with IPTG with an average yield (n= 2) of 0.7 mg/ml per liter of culture. A total of 1.162 mg/ml of recombinant antigen was obtained, and it is estimated that 3500 serologic samples may be tested with this amount of protein. Additional production experiments are being conducted to provide this recombinant antigen as much as needed by the onchocerciasis programs. | |||||
BibTeX:
@Poster{C.,
author = {Rodriguez Guadalupe C.},
title = {Expression of a recombinant antigen (Ov16) to Onchocerca volvulus},
booktitle = {Tuesday}
}
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Created by JabRef on 27/10/2007.