6 results on '"Stefania, Iametti"'
Search Results
2. Thermal unfolding of monomeric and dimeric β-lactoglobulins
- Author
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Alberto Schiraldi, Stefania Iametti, Francesco Bonomi, and Dimitrios Fessas
- Subjects
chemistry.chemical_compound ,Circular dichroism ,Crystallography ,Monomer ,Protein structure ,Differential scanning calorimetry ,chemistry ,Protein folding ,Thermal stability ,Calorimetry ,Biochemistry ,Fluorescence - Abstract
The thermal stabilities of dimeric bovine beta-lactoglobulin and monomeric equine beta-lactoglobulin were investigated at neutral pH by means of differential scanning calorimetry, circular dichroism, tryptophan fluorescence, and by binding of an hydrophobic probe. Differential scanning calorimetry showed the presence of two structural domains with different thermal stabilities in both proteins. Thermodynamic analysis of the calorimetric signal revealed that the two domains unfold independently according to a mechanism where an equilibrium step is followed by an irreversible transition. The spectroscopic data supported this model and allowed recognition of the structural regions corresponding to the more thermally stable domain. The differences in thermal stability between the two proteins can be primarily ascribed to the properties of the less stable domain.
- Published
- 2001
3. GroEL-assisted refolding of adrenodoxin during chemical cluster insertion
- Author
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Asya Grinberg, Stefania Iametti, Aloke Kumar Bera, Francesco Bonomi, Rita Bernhardt, and Giuseppe Vecchio
- Subjects
chemistry.chemical_classification ,biology ,Chemistry ,Sulfurtransferase ,GroES ,Rhodanese ,biology.organism_classification ,Biochemistry ,GroEL ,Enzyme ,Azotobacter vinelandii ,Adrenodoxin ,biology.protein ,bacteria ,Bovine serum albumin - Abstract
Chemical reconstitution of recombinant bovine adrenal mitochondrial apoadrenodoxin was carried out in the presence of the nonhomologous chaperone protein GroEL and of the cochaperone GroES, both in the presence and in the absence of ATP. The approach used here was different from the one characterizing studies on chaperone activity, as we used an adrenodoxin apoprotein, devoid of the cluster iron and sulfide, rather than a denaturant-unfolded form of the protein, and catalytic amounts of the chaperone proteins. A possible scaffolding role for two bacterial sulfurtransferases, namely, rhodanese from Azotobacter vinelandii and a rhodanese-like sulfurtransferase from Escherichia coli, was also investigated in the absence of the enzyme substrates. The extent and the rate of adrenodoxin refolding following cluster insertion was measured by spectroscopy and by monitoring the activity recovery in a NADPH–cytochrome c reduction assay. These measurements were carried out on the unresolved reaction mixture and on the adrenodoxin-containing fraction obtained by HPLC fractionation of the reconstitution mixture at different reaction times. The rate and extent of cluster insertion and activity recovery were substantially improved by addition of GroEL and increased with increasing the GroEL/apoadrenodoxin ratio. GroES and ATP had no effect by themselves, and did not enhance the effect of GroEL. A. vinelandii rhodanese, the E. coli sulfurtransferase, and bovine serum albumin had no effect on the rate and yield of chemical reconstitution. The accelerated chemical reconstitution of apoadrenoxin in the presence of GroEL is therefore attributable to a scaffolding effect of this protein.
- Published
- 2001
4. Reversible, Non-Denaturing Metal Substitution in Bovine Adrenodoxin and Spinach Ferredoxin and the Different Reactivities of [2Fe-2S]-Cluster-Containing Proteins
- Author
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Heike Uhlmann, Rita Bernhardt, Enzio Ragg, Stefania Iametti, Nicla Sala, and Francesco Bonomi
- Subjects
inorganic chemicals ,Circular dichroism ,Magnetic Resonance Spectroscopy ,Sulfide ,Cations, Divalent ,Molecular Sequence Data ,Iron–sulfur cluster ,Photochemistry ,Biochemistry ,Metal ,chemistry.chemical_compound ,Spinacia oleracea ,Adrenodoxin ,Animals ,Amino Acid Sequence ,Ferredoxin ,chemistry.chemical_classification ,biology ,Circular Dichroism ,Electron Spin Resonance Spectroscopy ,Nuclear magnetic resonance spectroscopy ,biology.organism_classification ,chemistry ,Metals ,Spectrophotometry ,visual_art ,visual_art.visual_art_medium ,Ferredoxins ,Spinach ,Cattle ,Oxidation-Reduction ,Cadmium - Abstract
The non-denaturing substitution of cluster iron by other metals was studied in spinach ferredoxin and in bovine adrenodoxin. Only some of several metal species tested (Cd2+, Zn2+, VO2+, Mn2+, Co2+, Ni2+) caused bleaching of the residual visible absorbance and of the EPR signals of the reduced ferredoxins. No formation of mixed-metal cluster was observed. The most reactive metal species were Cd2+ and Zn2+ and Cd2+ was found to react also with oxidized adrenodoxin. Metal-treated proteins were resolved into a mixture of apoprotein, metal-substituted protein and unreacted holoprotein. Their biological activity was proportional to the residual holoprotein concentration. Spinach ferredoxin and adrenodoxin were found to differ substantially with regard to their metal-substitution reactivity under oxidizing and reducing conditions, reaction time, and formation of apoprotein, which was more pronounced for spinach ferredoxin. Exchange of cluster iron with Cd2+ in adrenodoxin generated stable species containing 2 mol sulfide/mol protein and 2 or 5 mol cadmium/mol protein, respectively. The relative amount of the two substitution products depended on the experimental conditions. CD and NMR data on all the cadmium-substituted proteins suggest that iron replacement led to a significant structural rearrangement. Nevertheless, all the metal-substituted proteins could be re-converted into the native iron-containing form upon incubation with iron in the absence of reductants, of denaturing agents, and of an external source of sulfide. The different reactivity of the two proteins is discussed in terms of the cluster environment, along with the possible physiological relevance of these findings.
- Published
- 1996
5. Pro108 is important for folding and stabilization of adrenal ferredoxin, but does not influence the functional properties of the protein
- Author
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Heike Uhlmann, Rita Bernhardt, Stefania Iametti, Giuseppe Vecchio, and Francesco Bonomi
- Subjects
Iron-sulfur cluster assembly ,Models, Molecular ,endocrine system ,Protein Denaturation ,Protein Folding ,Proline ,Protein Conformation ,Mutant ,Gene Expression ,medicine.disease_cause ,Biochemistry ,Protein structure ,Adrenal Glands ,medicine ,Escherichia coli ,Animals ,Urea ,Denaturation (biochemistry) ,Ferredoxin ,Sequence Deletion ,Adrenal ferredoxin ,Chemistry ,Circular Dichroism ,Adrenodoxin ,Electron Spin Resonance Spectroscopy ,Temperature ,Recombinant Proteins ,Protein Structure, Tertiary ,Kinetics ,Spectrophotometry ,Protein folding ,Cattle ,Oxidation-Reduction - Abstract
The truncated mutant Met-adrenodoxin-(4-107)-peptide of bovine adrenal ferredoxin was expressed as apoprotein in Escherichia coli BL21 and could be reconstituted to the holoform by chemical or enzymatic methods. The reconstituted protein had spectroscopic, functional and redox properties similar to the Met-adrenodoxin-(4-108)-peptide of adrenal ferredoxin, into which the cluster was inserted upon expression in the same Escherichia coli strain. Rate of in vitro cluster insertion into the Met-adrenodoxin-(4-107) apoprotein was much lower than for the Met-adrenodoxin-(4-108) apoprotein under identical conditions. Comparative thermodynamic studies with the Met-adrenodoxin-(4-108)-peptide indicated that removal of Pro108 resulted in an extensive decrease of the overall stability of the protein in either oxidation state. The Met-adrenodoxin-(4-107)-peptide showed a higher sensitivity to urea denaturation and had a sensibly lower denaturation temperature, 44.8 degrees C, compared with 51.7 degrees C for mutant Met-adrenodoxin-(4-108). The stability of the reduced state of both mutants is slightly lower than that of the oxidized state indicating that this protein region does not undergo major structural changes upon reduction.
- Published
- 1997
6. Modifications occur at different structural levels during the heat denaturation of beta-lactoglobulin
- Author
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Stefania Iametti, Franco Bonomi, Giuseppe Vecchio, and B. De Gregori
- Subjects
Circular dichroism ,Protein Denaturation ,Hot Temperature ,biology ,Macromolecular Substances ,Dimer ,Circular Dichroism ,Sulfhydryl Reagents ,Lactoglobulins ,Biochemistry ,Dissociation (chemistry) ,Protein tertiary structure ,Protein Structure, Tertiary ,chemistry.chemical_compound ,Monomer ,chemistry ,Polymer chemistry ,biology.protein ,Iodoacetamide ,Organic chemistry ,Protein quaternary structure ,Beta-lactoglobulin - Abstract
Heat-induced modifications in the tertiary and quaternary structure of beta-lactoglobulin were followed at neutral pH for the protein at high temperature and for the protein that was heated and cooled. Fast changes in the environment of aromatic amino acids were apparent from near-ultraviolet-CD spectra of the heated protein and their intensity increased with increasing temperature. These modifications were irreversible only at temperatures higher than 65-70 degrees C. Addition of iodoacetamide during the heating/cooling cycle greatly reduced the extent of irreversible modification of the tertiary structure of the protein. Reaction of the native beta-lactoglobulin dimer with iodoacetamide or dithiobis(2-nitrobenzoic acid) was only observed upon heating at temperatures higher than 40 degrees C and resulted in progressive reaction of the unique sulfhydryl group in each of the two protein monomers. The sulfhydryl reagents induced release of a monomeric protein species that was no longer able to aggregate to the native dimeric form or to sequentially form polymers as found in the protein after heating at high temperature. Dimer dissociation was identified as the rate-limiting step in the reaction of beta-lactoglobulin with sulfhydryl reagents. It occurred at temperatures much lower than those required for appreciable modification of the tertiary structure of the protein, and had an extremely high activation energy (Ea = 213 kJ/mol). These results are compared with other published data, and a general mechanism for the formation of early reactive species in heat-treated beta-lactoglobulin at neutral pH is proposed which stresses the relevant role of a highly hydrophobic, molten-globule-like free monomer that has an exposed sulfhydryl group on its surface.
- Published
- 1996
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