Massimo Bellanda, Lorenzo Maso, Davide Doni, Marco Bortolus, Edith De Rosa, Federica Lunardi, Arianna Alfonsi, MartÃn Ezequiel Noguera, Maria Georgina Herrera, Javier Santos, Donatella Carbonera, Paola Costantini; Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2019, doi:10.1016/j.bbapap.2019.07.007.
n this work we combined different advanced spectroscopic analyses to explore the iron-binding properties of human frataxin, as isolated and at the FeS clusters assembly machinery. For the first time we used EPR spectroscopy to address this key issue providing clear evidence of the formation of a complex with a low symmetry coordination of the metal ion. By 2D NMR, we confirmed that iron can be bound in both oxidation states, a controversial issue, and, in addition, we were able to point out a transient interaction of frataxin with a N-terminal 6his-tagged variant of ISCU, the scaffold protein of the FeS clusters assembly machinery. To obtain insights on structure/function relationships relevant to understand the disease molecular mechanism(s), we extended our studies to four clinical frataxin mutants. All variants showed a moderate to strong impairment in their ability to activate the FeS cluster assembly machinery in vitro, while keeping the same iron-binding features of the wild type protein. This supports the multifunctional nature of frataxin and the complex biochemical consequences of its mutations.
Exploring iron-binding to human frataxin and to selected Friedreich ataxia mutants by means of NMR and EPR spectroscopies
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