title: Protein nanopore-based, single-molecule exploration of copper binding to an antimicrobial-derived, histidine-containing chimera peptide.
abstract: 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 α-HL protein as a powerful nanoscopic tool to probe Cu(2+)-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 Cu(2+)-bound peptide interaction to the α-HL pore. Combining single-molecule electrophysiology on reconstituted lipid membranes and fluorescence spectroscopy, we show that the association rate constant between the α-HL pore and a Cu(2+)-free peptide is higher than that of a Cu(2+)-complexed peptide. We posit that mainly due to conformational changes induced by the bound Cu(2+) 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 Cu(2+)-complexed peptide within the α-HL lumen region. The lower dissociation rate constant of the Cu(2+)-complexed peptide from α-HL pore, as compared to that of Cu(2+)-free peptide, supports the existence of a deeper free energy well for the protein interaction with a Cu(2+)-complexed peptide, which may be indicative of specific Cu(2+)-mediated contributions to the binding of the Cu(2+)-complexed peptide within the pore lumen.
authors.affiliation: Department of Physics, Laboratory of Molecular Biophysics and Medical Physics, Alexandru I. Cuza University, Blvd. Carol I, No. 11, Iasi 700506, Romania.
authors.forename: Loredana
authors.initials: L
authors.lastname: Mereuta
authors.forename: Irina
authors.initials: I
authors.lastname: Schiopu
authors.forename: Alina
authors.initials: A
authors.lastname: Asandei
authors.forename: Yoonkyung
authors.initials: Y
authors.lastname: Park
authors.forename: Kyung-Soo
authors.initials: KS
authors.lastname: Hahm
authors.forename: Tudor
authors.initials: T
authors.lastname: Luchian
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000737:chemistry; D001427:Bacterial Toxins / Q000737:chemistry*; D001665:Binding Sites; D003300:Copper / Q000737:chemistry*; D006460:Hemolysin Proteins / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D054806:Magainins / Q000737:chemistry; D008564:Membrane Potentials; D058608:Nanopores; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D023181:Antimicrobial Cationic Peptides; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D008051:Lipid Bilayers; D054806:Magainins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C021091:staphylococcal alpha-toxin; D006639:Histidine; D003300:Copper; C031161:cecropin A