Detecting metal-peptide complex formation by capillary electrophoresis

VIZIOLI, NM

Simposio; 23rd Latin-American Symposium on Biotechnology, Biomedical, Biopharmaceutical, and Industrial Applications of Capillary Electrophoresis and Microchip Technology; 2017

Throughout the evolution of organisms, about two dozen metal species have been selected to participate in biochemical reactions based on their physicochemical properties and bioavailability. On the other hand, some abiogenic metal species excluded from the evolutionary process, such as Hg2+, Pb2+, or Al3+ ions, upon entering the living organism, can affect the normal functioning of the cell by competing with the analogous metals for the respective protein binding sites. This is the basis of the leading hypothesis for the mechanism of the heavy metal toxicity postulating that the strange metal disrupts the structure of the host active site upon binding, which results in metalloprotein malfunction. However, substitution of natural metal cofactor by its abiogenic contender may generate beneficial effects on the host organism. Thus, gallium has been used to fight various forms of cancer, inflammatory, and infectious diseases. The explanation for this consists in the ability of Ga3+ cation to mimic ferric ion, Fe3+, interfering with the biological processes that require ferric cofactors. Even though a significant knowledge on the Ga3+-Fe3+ competition in biological systems has been accumulated, the mechanism of the process is still not well understood. To study the competition between Ga3+ and Fe3+, a model metal binding site has been selected from frataxin, an iron-binding protein. The putative iron-binding motif EEXXED corresponding to the first α-helical stretch of frataxin was grafted on a foreign peptide scaffold of the C-terminal α-helix from E. coli thioredoxin. Capillary electrophoresis (CE) together with circular dichroism, light scattering, isothermal titration calorimetry techniques, and computational calculations were applied to detect gallium-peptide complex formation. Experiments and simulations indicated that Ga3+interacts with the peptide through three side chains, inducing the α-helical conformation of the grafted motif. Furthermore, the involved side chains undergo significant conformational rearrangements upon binding. These results contribute to the knowledge of the metal-binding mechanisms in human frataxin.