Oligopeptide models of the metal binding loop of the bacterial copper efflux regulator protein CueR as potential Cu(I) chelators

Abstract Copper(I) binding features of oligopeptides, mimicking the metal binding loops (MBL) of the bacterial copper efflux regulator protein CueR, were investigated with an aim of exploring potential candidates for Cu(I)-sequestration. Two of the studied ligands comprise the MBL of E. coli CueR (Ac-ACPGDDSADCPI-NH 2 ; EC ) and V. cholerae CueR (Ac-SCPGDQGSDCPI-NH 2 ; VC ) while the third peptide is a His-containing variant of VC (Ac-SCHGDQGSDCSI-NH 2 ; HS ). UV-titrations of the ligands by Cu(I) at pH = 7.4 and monitoring the characteristic S Cys −  → Cu(I) charge transfer bands, together with CD-experiments, indicated the exclusive formation of monomeric Cu(I)-complexes (CuL) up to a 1:1 Cu(I):L ratio and the presence of oligometallic/cluster type complexes at Cu(I)-excess. Such a change in speciation and the domination of the latter species was also supported by comparing the ESI-MS spectra obtained at 0.9:1 and 2:1 Cu(I):peptide ratios. pH-dependence of the UV spectra of the Cu(I):peptide 1:1 complexes reflected the formation of the two thiolate-Cu(I) bonds well below neutral pH, as two successive deprotonation processes could be fitted by apparent p K a values falling in the pH range of 1.8–3.0 and 4.3–5.5. Cu(I)-binding affinity of the ligands was determined by competition experiments applying the bicinchoninate ion for the displacement of the Cu(I)-ligated peptides. The obtained conditional stability constants ( log β 11 pH 7.4  = 15.3, 15.8 and 16.3 for Cu( EC ), Cu( VC ) and Cu( HS ), respectively) indicate a strong affinity of Cu(I) to each ligand and suggest a slight destabilizing effect of charge repulsion between sidechain groups in EC while a possible Cu(I)-coordination of the histidine residue in HS .