1,720,975 research outputs found
Equilibrium partially folded states of B. licheniformis β -lactamase
No full text훽-Lactamases (penicillinases) facilitate bacterial resistance to antibiotics and are excellent theoretical and experimental models in protein structure, dynamics and evolution. Bacillus licheniformis exo-small penicillinase (ESP) is a Class A 훽 -lactamase with three tryptophan residues located one in each of its two domains and one in the interface between domains. The conformational landscape of three well-characterized ESP Trp→Phe mutants was characterized in equilibrium unfolding experiments by measuring tryptophan fuorescence, far-UV CD, activity, hydrodynamic radius, and limited proteolysis. The Trp→Phe substitutions had little impact on the native conformation, but changed the properties of the partially folded states populated at equilibrium. The results were interpreted in the framework of modern theories of protein folding.Fil: Risso, Valeria Alejandra. Universidad Nacional de Quilmes; Argentina. Universidad de Granada; EspañaFil: Ermacora, Mario Roberto. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentin
The structure of unliganded sterol carrier protein 2 from Yarrowia lipolytica unveils a mechanism for binding site occlusion
No full textIsolated or as a part of multidomain proteins, Sterol Carrier Protein 2 (SCP2) exhibits high affinity and broad specificity for different lipidic and hydrophobic compounds. A wealth of structural information on SCP2 domains in all forms of life is currently available; however, many aspects of its ligand binding activity are poorly understood. ylSCP2 is a well-characterized single domain SCP2 from the yeast Yarrowia lipolytica. Herein, we report the X-ray structure of unliganded ylSCP2 refined to 2.0 Å resolution. Comparison with the previously solved liganded ylSCP2 structure unveiled a novel mechanism for binding site occlusion. The liganded ylSCP2 binding site is a large cavity with a volume of more than 800 Å3. In unliganded ylSCP2 the binding site is reduced to about 140 Å3. The obliteration is caused by a swing movement of the C-terminal α helix 5 and a subtle compaction of helices 2–4. Previous pairwise comparisons were between homologous SCP2 domains with a uncertain binding status. The reported unliganded ylSCP2 structure allows for the first time a fully controlled comparative analysis of the conformational effects of ligand occupation dispelling several doubts regarding the architecture of SCP2 binding site.Fil: Gianotti, Alejo Román. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Klinke, 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: Ermacora, Mario Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentin
Binding properties of sterol carrier protein 2 (SCP2) characterized using Laurdan
Full text linkSterol carrier protein 2 (SCP2) binds lipids with high affinity and broad specificity. The overall hydrophobicity, fluidity, and dipolar dynamics of the binding site of SCP2 from Yarrowia lipolytica were characterized using the environmentally-sensitive fluorescent probe Laurdan. The study revealed a binding site with an overall polarity similar to that of dichloromethane and an internal phase comparable to that of phospholipid membranes with coexisting solid-ordered and liquid-crystalline states. The fluorescence properties of bound Laurdan also revealed that the binding site of SCP2 can accommodate competitively more than one ligand, with micro and nanomolar dissociation constants. The much higher affinity for the second than for the first ligand implies that the most prominent SCP2 species in the cellular context are those occupied by two ligands. Thus SCP2 may carry a highly populated lipid in the background and a second one, specific for the functional purpose of SCP2. Our findings are important for the characterization of SCP2 biological functions and the design of specific inhibitors.Fil: Gianotti, Alejo Román. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Ferreyra, Raul Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina. Universidad Nacional de Quilmes; ArgentinaFil: Ermacora, Mario Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentin
High-resolution structure of intramolecularly proteolyzed human mucin-1 SEA domain
No full textSEA domains are ubiquitous in large proteins associated with highly glycosylated environments. Certain SEA domains undergo intramolecular proteolysis involving a nucleophilic attack of a serine hydroxyl group on the preceding glycine carbonyl. The mucin-1 (MUC1) SEA domain has been extensively investigated as a model of intramolecular proteolysis. Since neither a general base, a general acid, nor an oxyanion hole could be identified in MUC1 SEA, it has been suggested that proteolysis is accelerated by a non-planarity of the scissile peptide bond imposed by protein folding. A reactant distorted peptide bond has been also invoked to explain the autoproteolysis of several unrelated proteins. However, the only evidence of peptide distortion in MUC1 SEA stems from molecular dynamic simulations of the reactant modeled upon a single NMR structure of the cleaved product. We report the first high-resolution X-ray structure of cleaved MUC1 SEA. Structural comparison with uncleaved SEA domains suggests that the number of residues evolutionarily inserted in the cleaved loop of MUC1 SEA precludes the formation of a properly hydrogen-bonded beta turn. By sequence analysis, we show that this conformational frustration is shared by all known cleaved SEA domains. In addition, alternative conformations of the uncleaved precursor could be modeled in which the scissile peptide bond is planar. The implications of these structures for autoproteolysis are discussed in the light of the previous research on autoproteolysis.Fil: Noguera, Martín Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Jakoncic, Jean. Brookhaven National Laboratory; Estados UnidosFil: Ermacora, Mario Roberto. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología - Universidad Nacional de Quilmes - GBEyB | Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología - Universidad Nacional de Quilmes - GBEyB | Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología - Universidad Nacional de Quilmes - GBEyB; Argentin
Folding of an Abridged β-Lactamase
Full text linkThe effects of C-terminal truncation on the equilibrium folding transitions and folding kinetics of B. licheniformis exo small β−lactamase (ES-βL) have been measured. ES-βL lacking 19 residues (ES-βLCΔ19) has no enzymic activity. Deletion of the last 14 residues produces ES-βLCΔ14, which is 0.1% active. The enzyme lacking nine residues (ES-βLCΔ9) is nearly fully active, has native optical and hydrodynamic properties, and is protease resistant, a distinguishing feature of the wild-type enzyme. Although ES-βLCΔ9 folds properly, it does so 4 orders of magnitude slower than ES-βL, making possible the isolation and characterization of a compact intermediate state (IP ES-βLCΔ9). Based on the analysis of folding rates and equilibrium constants, we propose that equilibrium between IP ES-βLCΔ9 and other intermediate slow folding. Residues removed in ES-βLCΔ9 and ES-βLCΔ14 are helical and firmly integrated into the enzyme body through many van der Waals interactions involving residues distant in sequence. The results suggest that the deleted residues play a key role in the folding process and also the existence of a modular organization of the protein matrix, at the subdomain level. The results are compared with other examples of this kind in the folding literature.Fil: Santos, Javier. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gebhard, Leopoldo German. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Risso, Valeria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Ferreyra, Raul Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Rossi, Juan Pablo Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Ermacora, Mario Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentin
Ultracompact states of native proteins
Full text linkA statistical analysis of circa 20,000 X-ray structures evidenced the effects of temperature of data collection on protein intramolecular distances and degree of compaction. Identical chains with data collected at cryogenic ultralow temperatures (≤160 K) showed a radius of gyration (Rg) significantly smaller than at moderate temperatures (≥240 K). Furthermore, the analysis revealed the existence of structures with a Rg significantly smaller than expected for cryogenic temperatures. In these ultracompact cases, the unusually small Rg could not be specifically attributed to any experimental parameter or crystal features. Ultracompaction involves most atoms and results in their displacement toward the center of the molecule. Ultracompact structures on average have significantly shorter van der Waals and hydrogen bonds than expected for ultralow temperature structures. In addition, the number of van der Waals contacts was larger in ultracompact than in ultralow temperature structures. The structure of these ultracompact states was analyzed in detail and the implication and possible causes of the phenomenon are discussed.Fil: Grille Coronel, Leandro. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Acierno, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Ermacora, Mario Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentin
Mapping the distribution of conformational information throughout a protein sequence
Full text linkThe three-dimensional structure of protein is encoded in the sequence, but many amino acid residues carry no essential conformational information, and the identity of those that are structure-determining is elusive. By circular permutation and terminal deletion, we produced and purified 25 Bacillus licheniformis β-lactamase (ESBL) variants that lack 5–21 contiguous residues each, and collectively have 82% of the sequence and 92% of the non-local atom–atom contacts eliminated. Circular dichroism and size-exclusion chromatography showed that most of the variants form conformationally heterogeneous mixtures, but by measuring catalytic constants, we found that all populate, to a greater or lesser extent, conformations with the essential features of the native fold. This suggests that no segment of the ESBL sequence is essential to the structure as a whole, which is congruent with the notion that local information and modular organization can impart most of the tertiary fold specificity and cooperativity.Fil: Gebhard, Leopoldo German. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Risso, Valeria Alejandra. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Santos, Javier. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ferreyra, Raul Gabriel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Noguera, Martín Ezequiel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ermacora, Mario Roberto. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin
X-ray evidence of a native state with increased compactness populated by tryptophan-less B. licheniformis β-lactamase
Full text linkβ-lactamases confer antibiotic resistance, one of the most serious world-wide health problems, and are an excellent theoretical and experimental model in the study of protein structure, dynamics and evolution. Bacillus licheniformis exo-small penicillinase (ESP) is a Class-A β-lactamase with three tryptophan residues located in the protein core. Here, we report the 1.7-Å resolution X-ray structure, catalytic parameters, and thermodynamic stability of ESPΔW, an engineered mutant of ESP in which phenylalanine replaces the wild-type tryptophan residues. The structure revealed no qualitative conformational changes compared with thirteen previously reported structures of B. licheniformis β-lactamases (RMSD = 0.4-1.2 Å). However, a closer scrutiny showed that the mutations result in an overall more compact structure, with most atoms shifted toward the geometric center of the molecule. Thus, ESPΔW has a significantly smaller radius of gyration (Rg) than the other B. licheniformis β-lactamases characterized so far. Indeed, ESPΔW has the smallest Rg among 126 Class-A β-lactamases in the Protein Data Bank (PDB). Other measures of compactness, like the number of atoms in fixed volumes and the number and average of noncovalent distances, confirmed the effect. ESPΔW proves that the compactness of the native state can be enhanced by protein engineering and establishes a new lower limit to the compactness of the Class-A β-lactamase fold. As the condensation achieved by the native state is a paramount notion in protein folding, this result may contribute to a better understanding of how the sequence determines the conformational variability and thermodynamic stability of a given fold.Fil: Risso, Valeria Alejandra. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Acierno, Juan Pablo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; ArgentinaFil: Capaldi, Stefano. Universita di Verona; ItaliaFil: Monaco, Hugo L.. Universita di Verona; ItaliaFil: Ermacora, Mario Roberto. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentin
X-ray structure of the mature ectodomain of phogrin
Full text linkPhogrin/IA-2 and ICA512/IA-2 are two paralogs receptor-type protein-tyrosine phosphatases (RPTP) that localize in secretory granules of various neuroendocrine cells. In pancreatic islet -cells, they participate in the regulation of insulin secretion, ensuring proper granulogenesis, and -cell proliferation. The role of their cytoplasmic tail has been partially unveiled, while that of their luminal region remains unclear. To advance the understanding of its structure?function relationship, the X-ray structure of the mature ectodomain of phogrin (ME phogrin) at pH 7.4 and 4.6 has been solved at 1.95- and 2.01-Å resolution, respectively. Similarly to the ME of ICA512, ME phogrin adopts a ferredoxin-like fold: a sheet of four antiparallel -strands packed against two -helices. Sequence conservation among vertebrates, plants and insects suggests that the structural similarity extends to all the receptor family. Crystallized ME phogrin is monomeric, in agreement with solution studies but in striking contrast with the behavior of homodimeric ME ICA512. The structural details that may cause the quaternary structure differences are analyzed. The results provide a basis for building models of the overall orientation and oligomerization state of the receptor in biological membranes.Fil: Noguera, Martín Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Primo, Maria Evangelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Jakoncic, Jean. Brookhaven National Laboratory; Estados UnidosFil: Poskus, Edgardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Solimena, Michele. Technische Universität Dresden.; Alemania. Max Planck Institute of Molecular Cell Biology and Genetics; AlemaniaFil: Ermacora, Mario Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biología Celular; Argentin
Land use intensification in the Rolling Pampa, Argentina: Diversifyingcrop sequences to increase yields and resource use
Full text linkIncreasing and maintaining high productivity levels presents a major challenge facing farmers today and will continue into the near future. More integrative and complex approaches to decision-making, besides adopting new technologies, are necessary for redesigning more productive, stable, and sustainable farming systems. Thus, novel crop sequences should be implemented to improve these properties of farming systems. The aim of our research was to characterize how different preceding crops that open recurrent sequences will impact on the productivity and resource use of the following crops, in order to determine the possibilities of increasing the frequency of double crops in rotations. Three field experiments were conducted under rainfed conditions at three sites in the Rolling Pampas of Argentina. The effects of seven cropping systems on the productivity of succeeding crops were evaluated at each location. The seven cropping systems included five double crops (rapeseed/soybean, wheat/soybean, barley/soybean, field pea/soybean, and field pea/maize) and two single crops (maize and soybean). The seven cropping systems were followed by the same crop sequence: wheat/soybean double crop and maize single crop in the first and second growing seasons, respectively. Radiation use and grain yield, water use and nitrogen uptake were evaluated for each crop in the sequence. Results indicate that repeating cereal crops in the cropping sequence reduces their productivities, while well balanced sequences that include legumes resulted in the highest productivities of cereal crops. Our findings highlight that diversifying cropping systems by adopting different double crops are practical options that can contribute to a more sustainable intensification of cropping systems specialized for grain crops. Increasing crop diversity in sequence influenced nitrogen uptake, among other factors, and may explain the enhanced crop yield in such systems. Our research highlights that crop diversification is critical in designing efficient and sustainable intensified crop sequences.Fil: Andrade, José Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura. Universidad de Buenos Aires. Facultad de Agronomía; ArgentinaFil: Poggio, Santiago Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura. Universidad de Buenos Aires. Facultad de Agronomía; ArgentinaFil: Ermacora, Mario Roberto. Asociación Argentina de Consorcios Regionales para la Experimentación Agrícola; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Satorre, Emilio Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura. Universidad de Buenos Aires. Facultad de Agronomía; Argentin
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