| Autor: |
Amadei M; Department of Biochemical Sciences 'A. Rossi Fanelli', Sapienza University of Rome, 00185 Rome, Italy., Niro A; Department of Biosciences and Territory, University of Molise, 86090 Pesche, Italy., Fullone MR; Department of Biochemical Sciences 'A. Rossi Fanelli', Sapienza University of Rome, 00185 Rome, Italy., Miele R; Department of Biochemical Sciences 'A. Rossi Fanelli', Sapienza University of Rome, 00185 Rome, Italy., Polticelli F; Department of Biology, University Roma Tre, 00146 Rome, Italy., Musci G; Department of Biosciences and Territory, University of Molise, 86090 Pesche, Italy., Bonaccorsi di Patti MC; Department of Biochemical Sciences 'A. Rossi Fanelli', Sapienza University of Rome, 00185 Rome, Italy. |
| Jazyk: |
angličtina |
| Zdroj: |
International journal of molecular sciences [Int J Mol Sci] 2023 Jul 25; Vol. 24 (15). Date of Electronic Publication: 2023 Jul 25. |
| DOI: |
10.3390/ijms241511919 |
| Abstrakt: |
Ferroportin (Fpn), a member of the major facilitator superfamily (MFS) of transporters, is the only known iron exporter found in mammals and plays a crucial role in regulating cellular and systemic iron levels. MFSs take on different conformational states during the transport cycle: inward open, occluded, and outward open. However, the precise molecular mechanism of iron translocation by Fpn remains unclear, with conflicting data proposing different models. In this work, amber codon suppression was employed to introduce dansylalanine (DA), an environment-sensitive fluorescent amino acid, into specific positions of human Fpn (V46, Y54, V161, Y331) predicted to undergo major conformational changes during metal translocation. The results obtained indicate that different mutants exhibit distinct fluorescence spectra depending on the position of the fluorophore within the Fpn structure, suggesting that different local environments can be probed. Cobalt titration experiments revealed fluorescence quenching and blue-shifts of λ max in Y54DA, V161DA, and Y331DA, while V46DA exhibited increased fluorescence and blue-shift of λ max . These observations suggest metal-induced conformational transitions, interpreted in terms of shifts from an outward-open to an occluded conformation. Our study highlights the potential of genetically incorporating DA into Fpn, enabling the investigation of conformational changes using fluorescence spectroscopy. This approach holds great promise for the study of the alternating access mechanism of Fpn and advancing our understanding of the molecular basis of iron transport. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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