Equilibrium unfolding of the PDZ domain of β2-syntrophin

b2-syntrophin, a dystrophin-associated protein, plays a pivotal role in insulin secretion by pancreatic b-cells. Itcontains a PDZ domain (b2S-PDZ) that, in complex with protein-tyrosine phosphatase ICA512, anchors the dense insulin gran-ules to actin filaments. The phosphorylation state of b2-syntrophin allosterically regulates the affinity of b2S-PDZ for ICA512,and the disruption of the complex triggers the mobilization of the insulin granule stores. Here, we investigate the thermal unfold-ing of b2S-PDZ at different pH and urea concentrations. Our results indicate that, unlike other PDZ domains, b2S-PDZ is margin-ally stable. Thermal denaturation experiments show broad transitions and cold denaturation, and a two-state model fit revealsa significant unfolded fraction under physiological conditions. Furthermore, Tm and Tmax denaturant-dependent shifts andnoncoincidence of melting curves monitored at different wavelengths suggest that two-state and three-state models fail toexplain the equilibrium data properly and are in better agreement with a downhill scenario. Its higher stability at pH >9 andthe results of molecular dynamics simulations indicate that this behavior of b2S-PDZ might be related to its charge distribution.All together, our results suggest a link between the conformational plasticity of the native ensemble of this PDZ domain and theregulation of insulin secretion.Fil: Torchio, Gabriela María. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Expresion y Plegamiento de Proteinas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ermacora, Mario Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Expresion y Plegamiento de Proteinas; ArgentinaFil: Sica, Mauricio Pablo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Expresion y Plegamiento de Proteinas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Equilibrium unfolding of the PDZ domain of β2-syntrophin

β2-syntrophin, a dystrophin-associated protein, plays a pivotal role in insulin secretion by pancreatic β-cells. It contains a PDZ domain (β2S-PDZ) that, in complex with protein-tyrosine phosphatase ICA512, anchors the dense insulin granules to actin filaments. The phosphorylation state of β2-syntrophin allosterically regulates the affinity of β2S-PDZ for ICA512, and the disruption of the complex triggers the mobilization of the insulin granule stores. Here, we investigate the thermal unfolding of β2S-PDZ at different pH and urea concentrations. Our results indicate that, unlike other PDZ domains, β2S-PDZ is marginally stable. Thermal denaturation experiments show broad transitions and cold denaturation, and a two-state model fit reveals a significant unfolded fraction under physiological conditions. Furthermore, Tm and Tmax denaturant-dependent shifts and noncoincidence of melting curves monitored at different wavelengths suggest that two-state and three-state models fail to explain the equilibrium data properly and are in better agreement with a downhill scenario. Its higher stability at pH > 9 and the results of molecular dynamics simulations indicate that this behavior of β2S-PDZ might be related to its charge distribution. All together, our results suggest a link between the conformational plasticity of the native ensemble of this PDZ domain and the regulation of insulin secretion.Instituto Multidisciplinario de Biología Celula

Composite cellulose fibre for affinity chromatography application

Affinity chromatographic supports is nowadays one of the most used and expensive consumable material for protein purification at the laboratory and industrial scale. The introduction of cost-effective materials is an important issue to address to spread the usefulness of this technology. Cotton fibres are a highly available natural material with excellent mechanical and structural properties which can be used for this propose. Nevertheless, fibre insolubility and low chemical reactivity are the major drawbacks to use this material for protein chromatography.In this work, a composite material prepared from polymethacrylate/cellulose fibres were used for the preparation of chromatographic materials containing immobilised proteins as adsorptive ligands. A green fluorescent protein and a protein A simil ligand containing cysteine in the terminal tags were immobilised onto the fibres by the epoxy-thiol chemistry. Buffer salt, pH, reaction time and pre-swelling procedure were optimised. Materials were characterised by fluorescence and electron microscopes, in addition to the binding capacity of the adsorbent materials.Fil: Carbajal, Maria Laura. 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; ArgentinaFil: Kikot, Pamela Alejandra. 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; ArgentinaFil: Torchio, Gabriela María. 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; ArgentinaFil: Fernandez Lahore, M.. Universitat Bremen. School of Enigineerring and Science Jacobs; AlemaniaFil: Grasselli, Mariano. 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

Equilibrium unfolding of the PDZ domain of β2-syntrophin

β2-syntrophin, a dystrophin-associated protein, plays a pivotal role in insulin secretion by pancreatic β-cells. It contains a PDZ domain (β2S-PDZ) that, in complex with protein-tyrosine phosphatase ICA512, anchors the dense insulin granules to actin filaments. The phosphorylation state of β2-syntrophin allosterically regulates the affinity of β2S-PDZ for ICA512, and the disruption of the complex triggers the mobilization of the insulin granule stores. Here, we investigate the thermal unfolding of β2S-PDZ at different pH and urea concentrations. Our results indicate that, unlike other PDZ domains, β2S-PDZ is marginally stable. Thermal denaturation experiments show broad transitions and cold denaturation, and a two-state model fit reveals a significant unfolded fraction under physiological conditions. Furthermore, Tm and Tmax denaturant-dependent shifts and noncoincidence of melting curves monitored at different wavelengths suggest that two-state and three-state models fail to explain the equilibrium data properly and are in better agreement with a downhill scenario. Its higher stability at pH > 9 and the results of molecular dynamics simulations indicate that this behavior of β2S-PDZ might be related to its charge distribution. All together, our results suggest a link between the conformational plasticity of the native ensemble of this PDZ domain and the regulation of insulin secretion.Instituto Multidisciplinario de Biología Celula