Harnessing the flexibility of peptidic scaffolds to control their copper(II)-coordination properties: A potentiometric and spectroscopic study

摘要

Designing small peptides that are capable of binding Cu~(2+) ions mainly through the side-chain functionalities is a hard task because the amide nitrogen atoms strongly compete for Cu~(2+) 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-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-Gly unit. A detailed potentiometric, mass spectrometric, and spectroscopic study (UV/Vis, CD, and EPR spectroscopy) showed that at a 1:1 Cu ~(2+)/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 Cu~(2+) ions with higher affinity. In addition, the calculated value of K_(eff) shows that this higher affinity for Cu~(2+) 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 Cu ~(2+) 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 Cu~(2+)-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 Cu ~(2+)-coordination properties of the C-Asp peptide, which binds Cu ~(2+) ions with higher affinity at all pH values, stabilizes the [CuHL]~(2+) species in a wider pH range, and has a slower Cu ~(2+)-exchange rate compared to O-Asp.