Metal ions binding exert a crucial influence upon the aggregation properties and stability of peptides, and the propensity of folding in various substates. Herein, we demonstrate the use of the alpha-HL protein as a powerful nanoscopic tool to probe Cu2+-triggered physicochemical changes of a 20 aminoacids long, antimicrobial-derived chimera peptide with a His residue as metal-binding site, and simultaneously dissect the kinetics of the free- and Cu2+-bound peptide interaction to the a-HL pore. Combining single-molecule electrophysiology on reconstituted lipid membranes and fluorescence spectroscopy, we show that the association rate constant between the alpha-HL pore and a Cu2+-free peptide is higher than that of a Cu2+-complexed peptide. We posit that mainly due to conformational changes induced by the Cu2+ on the peptide, the resulting complex encounters a higher energy barrier toward its association with the protein pore, stemming most likely from an extra entropy cost needed to fit the Cu2+-complexed peptide within the alpha-HL lumen region. The lower dissociation rate constant of the Cu2+-complexed peptide from alpha-HL pore, as compared to that of Cu2+-free peptide, supports the existence of a deeper free energy well for the protein interaction with a Cu2+-complexed peptide, which may be indicative of specific Cu2+-mediated contributions to the binding of the Cu2+-complexed peptide within the pore lumen. bound