Constant-pH MD Simulations Portray the Protonation and Structural Behavior of Four Decapeptides Designed to Coordinate Cu²⁺

The cyclic decapeptide C-Asp, containing one Asp residue and three His residues, was designed by Fragoso et al. (Chem. Eur. J. 2013, 19, 2076) to bind Cu²⁺ exclusively through the side chain groups and mimic copper coordination in metalloproteins. A variant of the cyclodecapeptide where Asp is substituted by Asn (C-Asn) has also been synthesized in addition to the linear (''open'') counterparts of both forms (O-Asp and O-Asn), testing the importance of cyclization and the presence of Asp in Cu²⁺ coordination (Chem. Eur. J. 2013, 19, 2076; Dalton Trans. 2013, 42, 6182). All peptides formed a major species at neutral pH that was able to coordinate Cu²⁺ exclusively through the neutral imidazole groups and the Asp side chain, when present, with C-Asp being the most effective. A detailed description of the protonation behavior of each histidine could help understanding the coordination species being formed in the pH range and eventually further optimizing the peptide's design. However, the standard current methods (NMR titrations) are not very suited for proximal groups titrating in the same pH range. In this work, we used the stochastic titration constant-pH molecular dynamics method to calculate the protonation curves and pK_a of each titrable residue in the four decapeptides, in the absence of Cu²⁺ ions. The global protonation curves obtained in our simulations are in very good agreement with the existing potentiometric titration curves. The histidines are titrating very closely, and the Asp forms abundant salt bridges with the basic residues, displaying an unusually low pK_a value. In addition, we could observe that the four peptides are very unstructured in the absence of copper, and not even the cyclic forms exhibit a significant β-sheet, unlike what could be expected from the presence of β-turn inducer units in this type of scaffold.