Harnessing the Flexibility of Peptidic Scaffolds to Control their Copper(II)-Coordination Properties: A Potentiometric and Spectroscopic Study

Authors

  • Ana Fragoso,

    1. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras (Portugal)
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  • Dr. Pedro Lamosa,

    1. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras (Portugal)
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  • Prof. Rita Delgado,

    1. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras (Portugal)
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  • Dr. Olga Iranzo

    Corresponding author
    1. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras (Portugal)
    • Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras (Portugal)
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Abstract

Designing small peptides that are capable of binding Cu2+ ions mainly through the side-chain functionalities is a hard task because the amide nitrogen atoms strongly compete for Cu2+ ion coordination. However, the design of such peptides is important for obtaining biomimetic small systems of metalloenyzmes as well as for the development of artificial systems. With this in mind, a cyclic decapeptide, C-Asp, which contained three His residues and one Asp residue, and its linear derivative, O-Asp, were synthesized. The C-Asp peptide has two Pro[BOND]Gly β-turn-inducer units and, as a result of cyclization, and as shown by CD spectroscopy, its backbone is constrained into a more defined conformation than O-Asp, which is linear and contains a single Pro[BOND]Gly unit. A detailed potentiometric, mass spectrometric, and spectroscopic study (UV/Vis, CD, and EPR spectroscopy) showed that at a 1:1 Cu2+/peptide ratio, both peptides formed a major [CuHL]2+ species in the pH range 5.0–7.5 (C-Asp) and 5.5–7.0 (O-Asp). The corrected stability constants of the protonated species (log K*CuH(O−Asp)=9.28 and log K*CuH(C−Asp)=10.79) indicate that the cyclic peptide binds Cu2+ ions with higher affinity. In addition, the calculated value of Keff shows that this higher affinity for Cu2+ ions prevails at all pH values, not only for a 1:1 ratio but even for a 2:1 ratio. The spectroscopic data of both [CuHL]2+ species are consistent with the exclusive coordination of Cu2+ ions by the side-chain functionalities of the three His residues and the Asp residue in a square-planar or square-pyramidal geometry. Nonetheless, although these data show that, upon metal coordination, both peptides adopt a similar fold, the larger conformational constraints that are present in the cyclic scaffold results in different behaviour for both [CuHL]2+ species. CD and NMR analysis revealed the formation of a more rigid structure and a slower Cu2+-exchange rate for [CuH(C-Asp)]2+ compared to [CuH(O-Asp]2+. This detailed comparative study shows that cyclization has a remarkable effect on the Cu2+-coordination properties of the C-Asp peptide, which binds Cu2+ ions with higher affinity at all pH values, stabilizes the [CuHL]2+ species in a wider pH range, and has a slower Cu2+-exchange rate compared to O-Asp.

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