Investigating the effect of proline residues on the coordination of divalent metal ions

The present study arises from the need to investigate the interaction of metal ions, such as Cu2+, Zn2+ and Fe2+, with short peptide sequences containing proline residues close to the metal binding site. Histidine imidazole usually acts as an anchoring site for Cu2+ already at acidic pH, afterward, increasing the alkalinity of the solution, the binding to ionized amide nitrogens can occur and it follows towards the N- or C-terminal direction with different extent of probability [1]. The occurrence of the same coordination behaviour for Zn2+ and Fe2+ is instead controversial [2, 3]. We studied three terminal protected short peptides: Ac-AAAHAAA-NH2, Ac-AAPHAAA-NH2 and Ac-AAPHPAA-NH2. In the second peptide, the Ala residue in position 3 has been replaced with a proline, while in the third peptide the two alanines adjacent to the histidine (position 3 and 5) have been replaced with prolines. The study allowed to investigate the coordination behaviour of the three metal ions under different pH values, with particular interest to the effect causes by the presence of proline residues close to the metal binding site. Indeed, prolines may provide greater structural rigidity to the peptide, hinder conformational changes and rearrangements around the metal center and make unavailable for binding their corresponding backbone amides. The characterization of the thermodynamics and speciation of the formed metal complexes has been achieved by means of potentiometry, UV-Vis spectrophotometry, circular dichroism, electron paramagnetic resonance and mass spectrometry. Financial support of the Polish National Science Centre (UMO-2020/37/N/ST4/03165) and of the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.1- NextGenerationEU (PRIN PNRR 2022- P2022EMY52) is gratefully acknowledge.