Publicação
Biophysical and Biochemical Characterization of Proteins involved in Transition Metals Homeostasis
| Resumo: | Cellular homeostasis depends on the coordinated activity of a vast array of proteins, including membrane transporters, metabolic enzymes or storage and protection proteins. The latter include the proteins from the Ferritin family of proteins, essential to the intracellular oxidative stress response, shielding the cell from harmful reactive oxygen species. DNA-binding proteins from starved cells (Dps), also termed miniferritins, protect bacterial DNA through two concomitant activities: indirect protection via ferroxidation and mineralization of up to 500 iron atoms and direct shielding through DNA binding and condensation. Their DNA-binding properties are related with their N-terminal tail extensions, of striking evolutionary diversity in length and composition. Given their importance to protein function, several questions remain unanswered regarding their conformational dynamics and functional features. The human mitochondrial ferritin (FtMt) was recently discovered as the mitochondrial counterpart to cytosolic ferritins. FtMt expression protects mitochondria (and the cell) from acute oxidative stress damage by regulating the levels of toxic labile iron. FtMt has been associated with several diseases related with iron and acute oxidative stress, but a deeper understanding of its biochemical and biophysical properties is still a relevant research goal. The work presented in this Thesis focused on probing the structural and functional properties of the Dps from the bacterium Deinoccocus grandis (DgrDps) and the human FtMt. The results from the DgrDps project reveal that the N-terminal tails of DgrDps may exist in different conformations, here investigated using Small Angle X-ray Scattering (SAXS), Dynamic Light Scattering (DLS) and Size-Exclusion Chromatography (SEC). Also, the position of tails can be modulated in vitro by external factors such as the ionic strength or the addition of specific transition metals. A novel iron-binding site was discovered and localized in the Nterminal tails using Mössbauer spectroscopy. The occupation of this binding site induces a conformational change in the tails, altering the structural properties of the protein. Regarding FtMt, it is shown that although the apo-form of FtMt is highly resistant, the accommodation of the iron mineral core leads to a decrease in the thermal stability of the protein and a particle aggregation effect correlated with the size of the iron core. |
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| Autores principais: | Guerra, João Pedro Leitão |
| Assunto: | DNA-binding protein from starved cells (Dps) Conformational dynamics of proteins; Mitochondrial Ferritin (FtMt) Synchrotron Radiation Circular Dichroism Small Angle X-ray Scattering Mössbauer spectroscopy |
| Ano: | 2022 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade Nova de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório Institucional da UNL |
| Resumo: | Cellular homeostasis depends on the coordinated activity of a vast array of proteins, including membrane transporters, metabolic enzymes or storage and protection proteins. The latter include the proteins from the Ferritin family of proteins, essential to the intracellular oxidative stress response, shielding the cell from harmful reactive oxygen species. DNA-binding proteins from starved cells (Dps), also termed miniferritins, protect bacterial DNA through two concomitant activities: indirect protection via ferroxidation and mineralization of up to 500 iron atoms and direct shielding through DNA binding and condensation. Their DNA-binding properties are related with their N-terminal tail extensions, of striking evolutionary diversity in length and composition. Given their importance to protein function, several questions remain unanswered regarding their conformational dynamics and functional features. The human mitochondrial ferritin (FtMt) was recently discovered as the mitochondrial counterpart to cytosolic ferritins. FtMt expression protects mitochondria (and the cell) from acute oxidative stress damage by regulating the levels of toxic labile iron. FtMt has been associated with several diseases related with iron and acute oxidative stress, but a deeper understanding of its biochemical and biophysical properties is still a relevant research goal. The work presented in this Thesis focused on probing the structural and functional properties of the Dps from the bacterium Deinoccocus grandis (DgrDps) and the human FtMt. The results from the DgrDps project reveal that the N-terminal tails of DgrDps may exist in different conformations, here investigated using Small Angle X-ray Scattering (SAXS), Dynamic Light Scattering (DLS) and Size-Exclusion Chromatography (SEC). Also, the position of tails can be modulated in vitro by external factors such as the ionic strength or the addition of specific transition metals. A novel iron-binding site was discovered and localized in the Nterminal tails using Mössbauer spectroscopy. The occupation of this binding site induces a conformational change in the tails, altering the structural properties of the protein. Regarding FtMt, it is shown that although the apo-form of FtMt is highly resistant, the accommodation of the iron mineral core leads to a decrease in the thermal stability of the protein and a particle aggregation effect correlated with the size of the iron core. |
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