Mechanism of DNA Recognition at a Viral Replication Origin

Recognition of the DNA origin by the Epstein-Barr nuclear antigen 1 (EBNA1) protein is the primary event in latentphase genome replication of the Epstein-Barr virus, a model for replication initiation in eukaryotes. We carried out an extensive thermodynamic and kinetic characterization of the binding mechanism of the DNA binding domain of EBNA1, EBNA1452-641, to a DNA fragment containing a single specific origin site. The interaction displays a binding energy of 12.7 kcal mol-1, with 11.9 kcal mol-1 coming from the enthalpic change with a minimal entropic contribution. Formation of the EBNA1452-641.DNA complex is accompanied by a heat capacity change of -1.22 kcal mol-1 K-1, a very large value considering the surface area buried, which we assign to an unusually apolar protein-DNA interface. Kinetic dissociation experiments, including fluorescence anisotropy and a continuous native electrophoretic mobility shift assay, confirmed that two EBNA1.DNA complex conformers are in slow equilibrium; one dissociates slowly (t1/2 approximately 41 min) through an undissociated intermediate species and the other corresponds to a fast twostep dissociation route (t1/2 approximately 0.8 min). In line with this, at least two parallel association events from two populations of protein conformers are observed, with on-rates of 0.25-1.6x10(8) m-1 s-1, which occur differentially either in excess protein or DNA molecules. Both parallel complexes undergo subsequent firstorder rearrangements of approximately 2.0 s-1 to yield two consolidated complexes. These parallel association and dissociation routes likely allow additional flexible regulatory events for site recognition depending on site availability according to nucleus environmental conditions, which may lock a final recognition event, dissociate and re-bind, or slide along the DNA.Fil: Oddo, Cristian. Fundación Instituto Leloir; ArgentinaFil: Freire Espeleta, Eleonora. Fundación Instituto Leloir; ArgentinaFil: Frappier, Lori. University of Toronto; CanadáFil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

The Endoplasmic Reticulum Glucosyltransferase Recognizes Nearly Native Glycoprotein Folding Intermediates

The UDP-Glc:glycoprotein glucosyltransferase (GT), a key player in the endoplasmic reticulum (ER) quality control of glycoprotein folding, only glucosylates glycoproteins displaying non-native conformations. To determine whether GT recognizes folding intermediates or irreparably misfolded species with nearly native structures, we generated and tested as GT substrates neoglycoprotein fragments derived from chymotrypsin inhibitor 2 (GCI2) bearing from 53 to 64 (full-length) amino acids. Fragment conformations mimicked the last stage-folding structures adopted by a glycoprotein entering the ER lumen. GT catalytic efficiency (V(max)/K(m)) remained constant from GCI2-(1-53) to GCI2-(1-58) and then steadily declined to reach a minimal value with GCI2-(1-64). The same parameter showed a direct hyperbolic relationship with solvent accessibility of the single Trp residue but only in fragments exposing hydrophobic amino acid patches. Mutations introduced (GCI2-(1-63)V63S and GCI2-(1-64)V63S) produced slight structural destabilizations but increased GT catalytic efficiency. This parameter presented an inverse exponential relationship with the free energy of unfolding of canonical and mutant fragments. Moreover, the catalytic efficiency showed a linear relationship with the fraction of unfolded species in water. It was concluded that the GT-derived quality control may be operative with nearly native conformers and that no alternative ER-retaining mechanisms are required when glycoproteins approach their proper folding.Fil: Caramelo, Julio Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Castro, Olga Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Parodi, Armando José A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

The Non-Structural NS1 Protein Unique to Respiratory Syncytial Virus: A Two-State Folding Monomer in Quasi-Equilibrium with a Stable Spherical Oligome

Human respiratory syncytial virus (hRSV) is a major infectious agent that cause pediatric respiratory disease worldwide. Considered one of the main virulence factors of hRSV, NS1 is known to suppress type I interferon response and signaling, thus favoring immune evasion. This, together with the fact that NS1 is unique to hRSV among paramyxoviruses, and that has no homology within databases, prompted us to investigate its conformational stability, equilibria and folding. Temperature cooperatively induces conformational changes leading to soluble spherical oligomers (NS1SOs) with amyloid-like or repetitive ß-sheet structures. The onset of the thermal transition is 45°C, and the oligomerization rate is increased by 25-fold from 40 to 46°C. Conformational stability analyzed by chemical perturbation of the NS1 monomer shows a two-state, highly reversible and cooperative unfolding, with a denaturant midpoint of 3.8 M, and a free energy change of 9.6±0.9 kcal⋅mol(-1). However, two transitions were observed in the chemical perturbation of NS1SOs: the first, from 2.0 to 3.0 M of denaturant, corresponds to a conformational transition and dissociation of the oligomers to the native monomer, indicating a substantial energy barrier. The second transition (2.0 to 3.5 M denaturant) corresponds to full unfolding of the native NS1 monomer. In addition, different cosolvent perturbations converged on the formation of ß-sheet enriched soluble oligomeric species, with secondary structure resembling those obtained after mild temperature treatment. Thus, a unique protein without homologs, structure or mechanistic information may switch between monomers and oligomers in conditions compatible with the cellular environment and be potentially modulated by crowding or compartmentalization. NS1 may act as a reservoir for increased levels and impact on protein turnover.Fil: Pretel, Miguel Esteban. Consejo Nacional de Invest.cientif.y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Invest.bioquimicas de Bs.as(i);Fil: Camporeale, Gabriela. Consejo Nacional de Invest.cientif.y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Invest.bioquimicas de Bs.as(i);Fil: de Prat Gay, Gonzalo. Consejo Nacional de Invest.cientif.y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Invest.bioquimicas de Bs.as(i)

Folding of a cyclin box: linking multitarget binding to marginal stability, oligomerization, and aggregation of the retinoblastoma tumor suppressor AB pocket domain

The retinoblastoma tumor suppressor (Rb) controls the proliferation, differentiation, and survival of cells in most eukaryotes with a role in the fate of stem cells. Its inactivation by mutation or oncogenic viruses is required for cellular transformation and eventually carcinogenesis. The high conservation of the Rb cyclin fold prompted us to investigate the link between conformational stability and ligand binding properties of the RbAB pocket domain. RbAB unfolding presents a three-state transition involving cooperative secondary and tertiary structure changes and a partially folded intermediate that can oligomerize. The first transition corresponds to unfolding of the metastable B subdomain containing the binding site for the LXCXE motif present in cellular and viral targets, and the second transition corresponds to the stable A subdomain. The low thermodynamic stability of RbAB translates into a propensity to rapidly oligomerize and aggregate at 37 °C (T50 = 28 min) that is suppressed by human papillomavirus E7 and E2F peptide ligands, suggesting that Rb is likely stabilized in vivo through binding to target proteins. We propose that marginal stability and associated oligomerization may be conserved for function as a "hub" protein, allowing the formation of multiprotein complexes, which could constitute a robust mechanism to retain its cell cycle regulatory role throughout evolution. Decreased stability and oligomerization are shared with the p53 tumor suppressor, suggesting a link between folding and function in these two essential cell regulators that are inactivated in most cancers and operate within multitarget signaling pathways.Fil: Chemes, Lucia Beatriz. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Noval, María Gabriela. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; ArgentinaFil: Sánchez Miguel, Ignacio Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: de Prat Gay, Gonzalo. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentin

Generation of sequence-specific, high affinity anti-DNA antibodies.

By taking advantage of the extreme stability of a protein-DNA complex, we have obtained two highly specific monoclonal antibodies against a predetermined palindromic DNA sequence corresponding to the binding site of the E2 transcriptional regulator of the human papillomavirus (HPV-16). The purified univalent antibody fragments bind to a double-stranded DNA oligonucleotide corresponding to the E2 binding site in solution with dissociation constants in the low and subnanomolar range. This affinity matches that of the natural DNA binding domain and is severalfold higher than the affinity of a homologous bovine E2 C-terminal domain (BPV-1) for the same DNA. These antibodies discriminate effectively among a number of double- and single-stranded synthetic DNAs with factors ranging from 125- to 20,000-fold the dissociation constant of the specific DNA sequence used in the immunogenic protein-DNA complex. Moreover, they are capable of fine specificity tuning, since they both bind less tightly to another HPV-16 E2 binding site, differing in only 1 base pair in a noncontact flexible region. Beyond the relevance of obtaining a specific anti-DNA response, these results provide a first glance at how DNA as an antigen is recognized specifically by an antibody. The accuracy of the spectroscopic method used for the binding analysis suggests that a detailed mechanistic analysis is attainable.Fil: Cerutti, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Centeno, Juan M.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Minute time scale prolyl isomerization governs antibody recognition of an intrinsically disordered immunodominant epitope

Conformational rearrangements in antibody·antigen recognition are essential events where kinetic discrimination of isomers expands the universe of combinations. We investigated the interaction mechanism of a monoclonal antibody, M1, raised against E7 from human papillomavirus, a prototypic viral oncoprotein and a model intrinsically disordered protein. The mapped 12-amino acid immunodominant epitope lies within a "hinge" region between the N-terminal intrinsically disordered and the C-terminal globular domains. Kinetic experiments show that despite being within an intrinsically disordered region, the hinge E7 epitope has at least two populations separated by a high energy barrier. Nuclear magnetic resonance traced the origin of this barrier to a very slow (t(1/2)∼4 min) trans-cis prolyl isomerization event involving changes in secondary structure. The less populated (10%) cis isomer is the binding-competent species, thus requiring the 90% of molecules in the trans configuration to isomerize before binding. The association rate for the cis isomer approaches 6 × 10(7) M(-1) s(-1), a ceiling for antigen-antibody interactions. Mutagenesis experiments showed that Pro-41 in E7Ep was required for both binding and isomerization. After a slow postbinding unimolecular rearrangement, a consolidated complex with K(D) = 1.2 × 10(-7) M is reached. Our results suggest that presentation of this viral epitope by the antigen-presenting cells would have to be "locked" in the cis conformation, in opposition to the most populated trans isomer, in order to select the specific antibody clone that goes through affinity and kinetic maturation.Fil: Fassolari, Marisol. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Chemes, Lucia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Gallo, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Smal, Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Sanchez, Ignacio E.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Development of a second generation prophylactic vaccine against human papillomavirus

Human papillomaviruses (HPV) are the etiologic agent for cervical cancer (CC), the second cause of cancer death in women worldwide. It is estimated that half a million new cases are diagnosed each year, mostly in developing countries due to the lack of massive programs for early detection of the virus. Recently, two prophylactic vaccines against the main oncogenic HPV types 16 and 18 (responsible for 80% of CC) have been introduced into market. Both of these vaccines, obtained as recombinants, have been shown to be safe and effective; however, their high cost works against its immediate impact in the incidence of HPV infection in developing and low-income countries. There is a need to have in hand second generation, low cost vaccines of massive use that will decrease CC cases in a large extent. With this in mind, we have developed a recombinant expression platform that allows us to obtain virus-like particles (VLPs) to formulate both effective and accessible vaccines against HPV infection.Human papillomaviruses (HPV) are the etiologic agent for cervical cancer (CC), the second cause of cancer death in women worldwide. It is estimated that half a million new cases are diagnosed each year, mostly in developing countries due to the lack of massive programs for early detection of the virus. Recently, two prophylactic vaccines against the main oncogenic HPV types 16 and 18 (responsible for 80% of CC) have been introduced into market. Both of these vaccines, obtained as recombinants, have been shown to be safe and effective; however, their high cost works against its immediate impact in the incidence of HPV infection in developing and low-income countries. There is a need to have in hand second generation, low cost vaccines of massive use that will decrease CC cases in a large extent. With this in mind, we have developed a recombinant expression platform that allows us to obtain virus-like particles (VLPs) to formulate both effective and accessible vaccines against HPV infection.Fil: Alonso, Leonardo Gabriel. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Xbio S.a; ArgentinaFil: Cerutti, Maria Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina. Xbio S.a; ArgentinaFil: Risso, Marikena Guadalupe. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Xbio S.a; ArgentinaFil: González, Mariángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentina. Xbio S.a; ArgentinaFil: Camporeale, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentin

Conformational Isomerization Involving Conserved Proline Residues Modulates Oligomerization of the NS1 Interferon Response Inhibitor from the Syncytial Respiratory Virus

Interferon response suppression by the respiratory syncytial virus relies on two unique nonstructural proteins, NS1 and NS2, that interact with cellular partners through high-order complexes. We hypothesized that two conserved proline residues, P81 and P67, participate in the conformational change leading to oligomerization. We found that the molecular dynamics of NS1 show a highly mobile C-terminal helix, which becomes rigid upon in silico replacement of P81. A soluble oligomerization pathway into regular spherical structures at low ionic strengths competes with an aggregation pathway at high ionic strengths with an increase in temperature. P81A requires higher temperatures to oligomerize and has a small positive effect on aggregation, while P67A is largely prone to aggregation. Chemical denaturation shows a first transition, involving a high fluorescence and ellipticity change corresponding to both a conformational change and substantial effects on the environment of its single tryptophan, that is strongly destabilized by P67A but stabilized by P81A. The subsequent global cooperative unfolding corresponding to the main β-sheet core is not affected by the proline mutations. Thus, a clear link exists between the effect of P81 and P67 on the stability of the first transition and oligomerization/aggregation. Interestingly, both P67 and P81 are located far away in space and sequence from the C-terminal helix, indicating a marked global structural dynamics. This provides a mechanism for modulating the oligomerization of NS1 by unfolding of a weak helix that exposes hydrophobic surfaces, linked to the participation of NS1 in multiprotein complexes.Fil: Conci, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Álvarez Paggi, Damián Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: de Oliveira, Guilherme A. P.. Universidade Federal do Rio de Janeiro; BrasilFil: Pagani, Talita Duarte. Universidade Federal do Rio de Janeiro; BrasilFil: Esperante, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Borkosky, Silvina Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Aran, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Alonso, Leonardo Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Mohana Borges, Ronaldo. Universidade Federal do Rio de Janeiro; BrasilFil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Influence des activités humaines sur la production et le stockage de matières à l'Holocène dans le bassin lacustre de Sarliève (Puy-de-Dôme, France)

L'objectif du programme est d'estimer l'impact des activités humaines sur le flux et le stockage de matières dans les bassins fluviatiles à l'Holocène, et en retour, d'évaluer l'impact des modifications anthropiques du milieu (sols et zones humides) sur ces activités. La méthode consiste à quantifier la matière (solide ou dissoute) prélevée aux versants à partir des stocks retenus dans les dépressions humides à différentes périodes de l'Holocène et en inférer des flux mis en relation avec le développement des sociétés

Thermodynamics of Cooperative DNA Recognition at a Replication Origin and Transcription Regulatory Site

Binding cooperativity guides the formation of protein-nucleic acid complexes, in particular those that are highly regulated such as replication origins and transcription sites. Using the DNA binding domain of the origin binding and transcriptional regulator protein E2 from human papillomavirus type 16 as model, and through isothermal titration calorimetry analysis, we determined a positive, entropy-driven cooperativity upon binding of the protein to its cognate tandem double E2 site. This cooperativity is associated with a change in DNA structure, where the overall B conformation is maintained. Two homologous E2 domains, those of HPV18 and HPV11, showed that the enthalpic-entropic components of the reaction and DNA deformation can diverge. Because the DNA binding helix is almost identical in the three domains, the differences must lie dispersed throughout this unique dimeric β-barrel fold. This is in surprising agreement with previous results for this domain, which revealed a strong coupling between global dynamics and DNA recognition.Fil: Dellarole, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: Sánchez Miguel, Ignacio Enrique. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; ArgentinaFil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. Fundación Instituto Leloir; Argentin